Therapeutic applications of p53 isoforms in regenerative medicine, aging and cancer

ABSTRACT

The present invention provides methods and compositions for modulating cell senescence and cell proliferation using isoforms of the p53 tumor suppressor protein. The methods and compositions of the invention find use in inhibiting cancer cell growth or in generating populations of cells for tissue regeneration through the modulation of cell senescence and proliferation.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/952,561, filed Jul. 26, 2013; which is a division of U.S. patentapplication Ser. No. 12/742,250, filed Sep. 29, 2010, which is a U.S.National Phase Application of International Application No. PCT/U.S.2008/080648 filed Oct. 21, 2008, which claims priority to U.S.Provisional Application Ser. No. 60/987,340, filed Nov. 12, 2007, thecontents of which are hereby incorporated by reference in the entiretyfor all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“77867-547300US-948913-SEQLIST.txt” created Jun. 26, 2015, andcontaining 6,424 bytes. The material contained in this text file isincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Cellular senescence was first described by Hayflick and Moorhead (1961)when they observed that normal human fibroblasts entered a state ofirreversible growth arrest after serial passage in vitro. In contrast,cancer cells did not enter this growth arrested state and proliferatedindefinitely. The maximum number of cell divisions that a cell canundergo, termed the Hayflick limit, varies from cell type to cell typeand organism. In fibroblasts, this number is about 50 divisions, afterwhich cell division ceases.

The process of cellular senescence can be triggered by multiplemechanisms, including telomere shortening, derepression of the INK4a/ARFlocus, and DNA damage. As discussed below, all three of these mechanismsimplicate the function of the tumor suppressor protein p53.

Telomere shortening provides a mechanism capable of counting celldivisions. Telomeres consist of repetitive DNA elements at the end oflinear chromosomes that protect chromosome ends from degradation andrecombination. Due to the intrinsic inability of the DNA replicationmachinery to copy the ends of linear molecules, telomeres becomeprogressively shorter with each round of replication, thus providing acounting mechanism for keeping track of the number of cell divisionsthat have occurred in a population of cells. As increasing numbers ofcell division occur, the telomeres reach a critically short length,which present as double-stranded DNA breaks that activate the p53 tumorsuppressor protein resulting in telomere-initiated senescence orapoptosis.

Derepression of the INK4a/ARF locus can also serve as a cell divisioncounting mechanism. The INK4a/ARF locus is normally expressed at verylow levels in most tissues of young organisms but progressively becomesderepressed with aging. Thus, a cell division counting mechanism isprovided by a progressively increased level of repression of theINK4a/ARF locus. The p16INK4a protein functions as an inhibitor ofcyclin-dependent kinases CDK4 and CDK6, thus providing a G1 cell cyclearrest. ARF regulates p53 stability through inactivation of thep53-degrading ubiquitin ligase MDM2.

The accumulation of DNA damage over time can also serve as a trigger forcell senescence. As an organism ages, increases in DNA mutations, DNAoxidation, and chromosome losses are observed. These observations haveprompted investigators to consider DNA damage as contributing tocellular senescence and organismal aging. As a guardian of cell cycleprogression after DNA damage, p53 plays a role here too, as p53 inducesthe expression of the cell cycle inhibitor p21 when a cell has undergoneDNA damage.

Given the direct impact that cell senescence has on cell division andcell cycle arrest, one would expect this process to play a central rolein such diverse processes as aging, cancer, and tissue regeneration. Thepresent invention provides methods and compositions for manipulatingthese diverse processes through the modulation of cell senescence.

BRIEF SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that theswitching of expression from one p53 isoform (Δ133p53) to another (p53β)results in replicative cellular senescence in normal human fibroblasts.Specifically, the present inventors have discovered that p53β andΔ133p53 promotes and inhibits, respectively, cellular senescence whenoverexpressed. siRNA-mediated knockdown of endogenous Δ133p53 inducedcellular senescence. Δ133p53 counteracted wild-type (wt) p53 to repressits transcriptional targets (p21^(WAF1) and miR-34a) and inhibit wtp53-mediated degradation of TRF2, allowing cell proliferation beyond thenormal senescence setpoint of telomeres. Accordingly, the presentinvention takes advantage of a novel telomere-mediated mechanism bywhich p53 regulates cellular senescence through inhibition of p53activity by its own natural isoforms.

Accordingly, in one aspect, the present invention provides a method ofpromoting senescence in a cell by contacting the cell with an agent thatinhibits the function or expression of Δ133p53, thereby promoting cellsenescence. The invention therefore also provides a use of such aninhibitory agent of Δ133p53 for manufacturing a medicament for treatinga disease in which cell senescence is inadequate.

In another aspect, the present invention provides a method of treatingor preventing cancer cell growth by promoting cell senescence bycontacting the cancer cell with an agent that inhibits the function orexpression of Δ133p53, thereby inhibiting cancer cell growth. Similarly,this invention provides a method for treating cancer by contactingcancer cells with an agent that inhibits the function or expression ofΔ133p53 in order to promote cancer cell senescence and therefore treatcancer. The invention therefore provides a use of such an inhibitoryagent of Δ133p53 for manufacturing a medicament for treating orpreventing a disease or condition involving undesirable cellularproliferation such as various types of cancer.

In this invention, an inhibitory agent of Δ133p53 may be an antisenseoligonucleotide, an siRNA (e.g., shRNA), a ribozyme, or a small organicmolecule. Preferably, such an inhibitor is effective specifically forthis particular isoform of p53 protein and not other isoforms.

In another aspect, the present invention provides a method of extendingthe replicative lifespan of a cell by inhibiting cell senescence bycontacting the cell with an agent that activates the function orexpression of Δ133p53, thereby inhibiting cell senescence and extendingthe replicative lifespan of the cell. The invention therefore provides ause of an activator of Δ133p53 for manufacturing a medicament fortreating a condition where cell replicative lifespan is inadequate.

In another aspect, the present invention provides a method of generatinga population of cells for tissue regeneration by inhibiting cellsenescence by: (a) contacting a cell suitable for tissue regenerationthat has a finite number of cell divisions with an agent that activatesthe function or expression of Δ133p53, thereby inhibiting cellsenescence and increasing the number of cell divisions undergone by thecell, and (b) culturing the cell to obtain a cell population, therebygenerating a population of cells for tissue regeneration. In someaspects of this embodiment, the agent comprises a nucleic acid for theoverexpression of Δ133p53, such as a polynucleotide sequence (e.g., aDNA sequence) encoding Δ133p53 or an expression cassette capable ofoverexpressing the protein. The method for producing cell populationsfor tissue regeneration can be useful for treating or preventingdegenerative diseases including various age-related conditions such asosteoporosis, osteoarthritis, macular degeneration, and atherosclerosis.

In another aspect, the present invention provides a method of promotingsenescence in a cell by contacting the cell with an agent that activatesthe function or expression of p53β, thereby promoting cell senescence.In some embodiments, the agent comprises a nucleic acid encoding p53 β,such as a polynucleotide sequence (e.g., a DNA sequence) or expressioncassette encoding and capable of overexpressing p53β protein.

In another aspect, the present invention provides a method of treatingor preventing cancer cell growth by promoting cell senescence bycontacting the cancer cell with an agent that activates the function orexpression of p53 β, thereby inhibiting cancer cell growth. In someembodiments, the agent comprises a nucleic acid encoding p53β, such as apolynucleotide sequence (e.g., DNA) or expression cassette encoding andcapable of overexpressing p53β protein.

In another aspect, the present invention provides a method of extendingthe replicative lifespan of a cell by inhibiting cell senescence, themethod comprising the step of contacting the cell with an agent thatinhibits the function or expression of p53β, thereby inhibiting cellsenescence and extending the replicative lifespan of the cell.Similarly, the invention provides a method of preventing or treating adegenerative disease by inhibiting cell senescence. Degenerativediseases include various age-related conditions such as osteoporosis,osteoarthritis, macular degeneration, and atherosclerosis. For instance,the method includes the step of contacting cells or tissues that aresusceptible of the degenerative disease or involved in the disease withan agent that inhibits the function or expression of p53 β, thereforeinhibits cell senescence and prevents or treats the degenerativedisease.

In another aspect, the present invention provides a method of extendingthe replicative lifespan of a cell by inhibiting cell senescence by wayof contacting the cell with an agent that inhibits the function orexpression of miR-34a, thereby inhibiting cell senescence and extendingthe replicative lifespan of the cell. An exemplary agent useful for thispurpose is an antisense oligonucleotide that specifically inactivatesmiR-34a.

In another aspect, this invention provides a method for enhancing orrestoring immune functions by extending T cell lifespan. The methodincludes the step of contacting the T cell with an agent that activatesthe function or expression of Δ133p53, thereby extending the lifespan ofthe T cell and enhancing or restoring immune functions. The agent maycomprise a polynucleotide sequence encoding Δ133p53, or comprise anexpression cassette comprising a polynucleotide sequence encodingΔ133p53. In contrast, the invention also provides a method for enhancingor restoring immune functions by extending T cell lifespan by the meansof contacting the T cell with an agent that inhibits the function orexpression of p53β. Such an agent may be an siRNA, e.g., an shRNA, or aribozyme. Furthermore, a method is provided for enhancing or restoringimmune functions by extending T cell lifespan, the method comprising thestep of contacting the T cell with an agent that inhibits the functionor expression of miR-34a (such as an antisense oligonucleotide capableof inactivating miR-34a), thereby extending the lifespan of the T celland enhancing or restoring immune functions.

In another aspect, the present invention provides a method of generatinga population of cells for tissue regeneration by inhibiting cellsenescence by: (a) contacting a cell suitable for tissue regenerationthat has a finite number of cell divisions with an agent that inhibitsthe function or expression of p53β, thereby inhibiting cell senescenceand increasing the number of cell divisions undergone by the cell, and(b) culturing the cell to obtain a cell population, thereby generating apopulation of cells for tissue regeneration.

In this invention, an inhibitory agent of p53β may be an antisenseoligonucleotide, an siRNA (e.g., shRNA), a ribozyme, or a small organicmolecule. Preferably, such an inhibitor is effective specifically to oneisoform of the p53 protein and not to other isoforms.

In yet another aspect, the present invention provides a composition forpromoting cell senescence comprising an siRNA directed to Δ133p53. Insome cases, the siRNA is an shRNA. In an aspect of this embodiment, thesiRNA comprises or consists of the sequence 5′-UGU UCA CUU GUG CCC UGACUU UCA A-3′ (SEQ ID NO:1) or 5′-CUU GUG CCC UGA CUU UCA A[dT][dT]-3′(SEQ ID NO:2). Optionally, a physiologically acceptable excipient isalso present in this composition. In one example, this composition maybe used for promoting senescence and inhibiting cellular proliferationby suppressing Δ133p53 activity, and therefore for use in treatingconditions relevant to undesired cell proliferation, such as varioustypes of cancer.

In another aspect, the present invention provides a method foridentifying a compound that modulates cell senescence via its effect onΔ133p53 or p53β. In general, the method includes these steps: (a)contacting a candidate compound with a sample that comprises Δ133p53 orp53β, and (b) determining the functional effect of the compound (such asincreased or decreased cell proliferation, cell cycle arrest, orapoptosis), based on which one may determine whether the compound is amodulator (e.g., an activator or inhibitor) of the respective p53isoform. For instance, increased cell proliferation would indicate atest compound's role as a suppressor of senescence; whereas decreasedcell proliferation, cell cycle arrest, or increased apoptosis wouldindicate a test compound's role as a promoter of the senescence.Accordingly, the identified modulator may be useful for preventing ortreating cancer, or for extending a cell's replicative lifespan,depending on the specific effect of the modulator on Δ133p53 or p533 andtherefore on senescence. A candidate compound can be of any chemicalnature: a small molecule or a macromolecule such as protein, lipid,polysaccharide, polynucleotide, etc., synthetic or naturally occurring.

In various aspects of this invention, the agent useful for suppressingthe effects of a p53 isoform by inhibiting or inactivating theexpression or function of the isoform can be an antisenseoligonucleotide, an siRNA (such as a shRNA), a ribozyme, or a smallorganic molecule. In further aspects, the cell whose growth is to besuppressed can be a cancer cell.

In some aspects of the above embodiments, the agent useful for enhancingthe effects of a p53 isoform comprises a DNA for the overexpression ofΔ133p53 or p53β.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1. p53β and Δ133p53 are involved in cellular senescence andproliferation: (A) Induction of p53β and repression of Δ133p53 atreplicative senescence. The immunoblot analyses were performed inearly-passage (Y) and senescent (S) human fibroblast strains MRC-5 andWI-38. The examined passage numbers were 30 (Y) and 65 (S) for MRC-5;and 30 (Y) and 58 (S) for WI-38. TLQ40, an antibody detecting p53βisoforms; MAP4, an antibody detecting Δ133p53; CM1, an antibodydetecting wt p53. Δ40p53β (Ghosh, A. et al., Mol. Cell. Biol. 24:7987(2004)) was a predominant form detected by TLQ40 and was constitutivelyexpressed in both early-passage and senescent cells. p21^(WAF1)expression was also examined β-actin was a loading control. H1299 cellsoverexpressing p53β and CC1 cells (Horikawa, I. et al., Hum. Mol. Genet.4:313 (1995)) were used as the positive controls for p53β and Δ133p53,respectively. (B) miR-34a expression during replicative senescence. Thesame set of MRC-5 and WI-38 fibroblasts as used in (A) were examined formiR-34a expression by real-time qRT-PCR. The data were normalized withcontrol RNU66 expression and shown as the relative values. Threeindependent experiments were carried out and the reproducible resultswere obtained. (C) Retroviral overexpression of p53β and Δ133p53 inhuman fibroblasts. The retroviral vectors driving wt p53, FLAG-taggedp53β and FLAG-tagged Δ133p53 were transduced to human fibroblasts atearly passage (at passage number 30 for both strains) and the immunoblotanalyses of the overexpressed p53 isoforms, MDM2 and p21^(WAF1) wereperformed. Protein samples were prepared from the cells at 8 days afterretroviral transduction. The anti FLAG antibody detected FLAG-taggedp53β and FLAG-tagged Δ133p53, and the DO-12 antibody detected all thethree p53 isoforms. β-actin was a loading control. (D) Effects of p53βand Δ133p53 on cell proliferation. The cells were plated at 8 days afterretroviral transduction and the cell numbers were counted daily. Vector(open squares), wt p53 (open diamonds), FLAG-p53β (closed circles), andFLAG-Δ133p53 (closed triangles). The data (mean ±standard error) werefrom three independent experiments. (E) Senescence-associatedβ-galactosidase (SA-β-gal) assay. The cells were examined at 8 daysafter retroviral transduction. The data (mean±standard error) were fromthree independent experiments.

FIG. 2. Overexpression of Δ133p53 extends replicative lifespan. (A)Examination of cellular replicative lifespan. The FLAG-Δ133p53retroviral vector (open circles) or the control vector (open squares)was transduced to human fibroblasts at late passage (MRC-5 at passage 53and WI-38 at passage 51). The cumulative population doublings (PDL) werecalculated and plotted to days after G418 selection. (B) Telomere lengthand telomeric 3′ overhang in Δ133p53-overexpressing cells. Genomic DNAsamples from MRC-5 with FLAG-Δ133p53 or control vector were used in thein-gel hybridization with ³²P-[CCCTAA]₄ (SEQ ID NO:5) probe underdenatured (for telomere length) and native (for telomeric 3′ overhang)conditions. Lane 1, MRC-5 before transduction; lanes 2-3, vector control(days 4 and 35 post selection); lanes 4-6, FLAG-Δ133p53 (days 4, 35 and96 post selection). The telomere lengths were measured as peak TRF(terminal restriction fragment) lengths. The amounts of telomeric 3′overhang were normalized with loaded DNA amounts (EtBr) and shown aspercent signals to the cells before transduction. (C) Repression ofmiR-34a expression by Δ133p53. RNA samples from MRC-5 (at passage 53)before transduction (day 0), MRC-5 with control vector and MRC-5overexpressing Δ133p53 (at days 20, 36 and 96 post selection) wereanalyzed as in FIG. 1B. The value before transduction was defined as 1.0and the expression levels in the other samples were expressed as therelative values. (D) Extension of cellular replicative lifespan byinhibition of miR-34a expression. The late-passage MRC-5 fibroblastswere transfected with the antisense oligonucleotide against miR-34a andthe control oligonucleotide (EGFP) every four days and the cumulativePDL were examined as in (A) (left panel). The downregulation of miR-34aexpression was confirmed by the real-time qRT-PCR (right panel).

FIG. 3. Knockdown of endogenous Δ133p53 expression induces cellularsenescence. Early-passage MRC-5 fibroblasts (at passage 32) weretransfected with siRNAs targeting Δ133p53 (Δ133si-1 and Δ133si-2) and acontrol oligonucleotide twice (at day 1 and day 4), and at day 7 wereused for immunoblot analyses (A) and examined for SA-(3-Gal activity (Band C) and bromo-deoxyuridine (BrdU) incorporation (D). (A)siRNA-mediated repression of Δ133p53. Expressions of wt p53 (DO-1antibody), Δ133p53 (MAP4 antibody) and p21^(WAF1) were examined β-actinwas a loading control. (B) Representative pictures of SA-β-Gal staining.(C) Summary of SA-β-Gal assay. The data (mean±standard error) were fromtwo independent experiments. (D) Summary of BrdU incorporation assay.The number of BrdU-positive cells/the total number of cells examined (atleast 100 cells for each sample) was recorded.

FIG. 4. Δ133p53 inhibits wt p53-mediated degradation of TRF2. (A)Immunoblot analysis of TRF2 expression in Δ133p53-overexpressing cells.MRC-5 and WI-38 fibroblasts with FLAG-Δ133p53 or control vector atindicated days after G418 selection (see FIG. 2A) were examined for TRF2expression. The expression of Δ133p53 was confirmed by anti-FLAGantibody. (B) Immunoblot analysis of TRF2 in p53-knocked down cells. Atelomerase-immortalized fibroblast cell line (hTERT/NHF) was transducedwith the retroviral shRNA vector targeting p53. (C) p53 regulation ofmiR-34a expression. hTERT/NHF cells transduced with p53 shRNA (left) andtreated with 10 μM of Nutlin-3a for 36 h (right) were examined formiR-34a expression, as in FIG. 1B. The data is shown as the relativeexpression level to control cells (−). (D) TRF2 expression infibroblasts from Li-Fraumeni syndrome patients. MDAH041 has a p53frame-shift mutation (−), and MDAH087 and MDAH172 have p53 missensemutations (mt) (Yin, Y. et al.,. Cell 70:937 (1992)). p53-heterozygous(wt/− and wt/mt) and homozygous (−/− and mt/mt) fibroblasts wereexamined in parallel. (E) Δ133p53 abrogates wt p53-mediateddownregulation of TRF2. Cells (293T) were retrovirally transduced withMyc-tagged TRF2, wt p53 and FLAG-tagged Δ133p53 as indicated. Anti-Myc,anti-FLAG and DO-1 antibodies were used in immunoblot analyses. (F)Effects of a proteasome inhibitor (MG-132) on TRF2 expression. ControlhTERT/NHF, Δ133p53-overexpressing hTERT/NHF and p53-knocked downhTERT/NHF were cultured in the presence (+) or absence (−) of 10 μM ofMG-132 (Sigma-Aldrich) for 5 hrs and examined for TRF2 expression. (G)TRF2 accumulation by the inhibition of Siah-1A activity. TheFLAG-tagged, dominant-negative mutant of Siah-1A (FLAG-Siahl-ARING) wasexpressed in MDAH041 fibroblasts (arrow). β-catenin, known to bedegraded by Siah-1A (Matsuzawa, S. I. et al., Mol. Cell 7:915 (2001)),was examined to confirm the activity of FLAG-Siahl-ΔARING. β-actin was aloading control in (A), (B), (D), (E), (F) and (G). (H) Overexpressionof TRF2 extends replicative lifespan. MRC-5 fibroblasts at passage 39were transduced with the retroviral vector driving A133p53 or controlvector (G418 resistant) and selected with G418 for 7 days. These cellswere then transduced with the retroviral vector driving TRF2 or controlvector (puromycin resistant), selected with puromycin, and examined forcellular replicative lifespan as in FIG. 2A. For each combination ofretroviral transductions, the cumulative PDL at days post puromycinselection were recorded.

FIG. 5. MAP4 specifically recognizes Δ133p53. H1299 cells (p53-null)transfected with the expression vector for wild-type (wt) p53, p53β orA133p53 were analyzed in Western blot using MAP4 (left) and DO-1 (right)antibodies. MAP4 detects Δ133p53, but not wt p53 or p53 β.

FIG. 6. mRNA expression analysis of p53 isoforms in human fibroblasts.The same sets of cells as in FIG. 1A were analyzed by RT-PCR. Incontrast to protein levels, mRNA of p53β was decreased in senescentcells and Δ133p53 was primarily unchanged. The primers to amplify wt p53were: 5′-CTC ACC ATC ATC ACA CTG GAA-3′ (SEQ ID NO:6) and 5′-TCA TTC AGCTCT CGG AAC ATC-3′ (SEQ ID NO:7). The primers specifically detecting thealternative splicing for p53β were: 5′-CTT TGA GGT GCG TGT TTG TGC-3′(SEQ ID NO:8) and 5′-TTG AAA GCT GGT CTG GTC CTG A-3′ (SEQ ID NO:9). Theprimers specifically amplifying Δ133p53 mRNA transcribed from thepromoter in intron 4 were: 5′-TGG GTT GCA GGA GGT GCT TAC-3′ (SEQ IDNO:10) and 5′-CCA CTC GGA TAA GAT GCT GAG G-3′ (SEQ ID NO:11). The lowerbands correspond to the reported Δ133p53 sequences (GenBank DQ186650).The upper bands are from mRNA with intron 5 unspliced. GAPDH wasamplified as a control as previously described (Horikawa, I. et al.,Mol. Carcinog. 22:65 (1998)).

FIG. 7. Senescence-associated (SA)-β-galactosidase (gal) staining ofMRC-5 fibroblasts overexpressing wt p53, FLAG-tagged p53β andFLAG-tagged Δ133p53. MRC-5 with control vector is also shown.

FIG. 8. p53β overexpression induces cellular senescence in humanfibroblasts with ectopically expressed telomerase. (A) Effects of p53βon cell proliferation. hTERT (human telomerase reversetranscriptase)-immortalized human fibroblasts (hTERT/NHF) weretransduced with the retroviral vector driving FLAG-tagged p53β orcontrol vector (a zeocin-resistant version). Cell proliferation assaywas carried out as in FIG. 2B. (B) Upregulation of p21^(WAF1) by p53βoverexpression in hTERT/NHF cells. (C) Representative pictures ofSA-β-gal staining. (D) Summary of SA-β-gal staining. The data weremean±standard error from three independent experiments.

FIG. 9. Δ133p53 overexpression delays replicative senescence inlate-passage human fibroblasts. MRC-5 fibroblasts with control vector orFLAG-tagged Δ133p53 (same cells as in FIG. 3A) were stained for SA-β-galactivity at 10 days post G418 selection. (A) Representative pictures.(B) Data summary.

FIG. 10. Knockdown of endogenous Δ133p53 induces cellular senescence.Early-passage WI-38 fibroblasts (at passage 30) were transfected withsiRNAs targeting Δ133p53 (Δ133si-1 and Δ133si-2) and a controloligonucleotide and examined in immunoblot analyses (A), SA-β-Gal assay(B) and BrdU incorporation assay (C), as performed in FIG. 3.

FIG. 11. Nutlin-3A downregulates TRF2 protein in a p53-dependent manner.hTERT/NHF cells with (+) or without (−) p53 shRNA were treated with 10μM of Nutlin-3A (Cayman Chemical) for the indicated time period andexamined for TRF2, p53 and MDM2 amounts in immunoblot analyses. β-actinwas a loading control.

FIG. 12. The p53 knockdown-induced increase in TRF2 protein is not dueto an increase in TRF2 mRNA. hTERT/NHF cells with (+) and without (−)p53 shRNA were examined for TRF2 mRNA expression by the real-timeqRT-PCR (cat. no. 04689038001, Roche Applied Science).

FIG. 13. Δ133p53 does not affect TRF2 expression in the absence of wtp53. (A) wt p53, FLAG-tagged p53β and FLAG-tagged Δ133p53 wereretrovirally expressed in MDAH041 (p53−/−) fibroblasts. Neither p53β norΔ133p53 changed TRF2 expression in these cells, while a significantdecrease in TRF2 was observed with wt p53. The expression of Siah-1A wasalso examined and shown to be induced by wt p53. The expression of p53isoforms was confirmed with anti-FLAG antibody and/or anti-p53 antibody(DO-1). (B) Downregulation of TRF2 by Siah-1A overexpression. Cells(293T) were retrovirally transduced with Myc-tagged TRF2, FLAG-taggedSiahl-Δ6 (a stable form of Siah-1A) (Tanikawa, J. et al., J. Biol. Chem.279:55393 (2004)) and wt 53 as indicated. Anti-Myc, anti-FLAG and DO-1antibodies were used in immunoblot analyses. (C) Δ133p53 was knockeddown by p53 shRNA in CC1 cells, which express Δ133p53 but not wt p53 dueto a genomic rearrangement (Horikawa, I. et al., Hum. Mol. Genet. 4:313(1995)). No change in TRF2 expression was observed with a remarkabledecrease in Δ133p53 (confirmed by immunoblot using MAP4). β-actin was aloading control in (A), (B) and (C).

FIG. 14. Replicative senescence-associated changes in expression ofendogenous p53 isoforms and p53-regulated microRNA-34a. a, Induction ofp53β and repression of Δ133p53 at replicative senescence. The immunoblotanalyses were performed in early-passage (Y) and senescent (S) humanfibroblast strains MRC-5 and WI-38. The examined passage numbers were 30(Y) and 65 (S) for MRC-5; and 30 (Y) and 58 (S) for WI-38. TLQ40, anantibody detecting p53β isoforms; MAP4, an antibody detecting Δ133p53;DO-12, an antibody used to detect full-length p53; CM1, an antibody usedto simultaneously detect full-length p53, p53β and Δ133p53. Δ40p53β wasa predominant form detected by TLQ40 and was constitutively expressed inboth early-passage and senescent cells. p21^(WAF1) expression was alsoexamined β-actin was a loading control. H1299 cells overexpressing p53βand CC1 cells were used as the positive controls for p53β and Δ133p53,respectively. b, miR-34a expression during replicative senescence. Thesame set of MRC-5 and WI-38 fibroblasts as used in a were examined formiR-34a expression by real-time qRT-PCR. The data were normalized withcontrol RNU66 expression and shown as the relative values (mean±s.d.from triplicate sample). Three independent experiments were carried outand the reproducible results were obtained. *, p<0.001. **, p<0.01. cand d, Extension of cellular replicative lifespan by the inhibition ofmiR-34a expression. Late-passage MRC-5 fibroblasts (at passage 58) weretransfected with the antisense oligonucleotide against miR-34a and thecontrol oligonucleotide (EGFP). The effectiveness of the antisensemiR-34a was confirmed by the real-time qRT-PCR (error bars represents.d. from triplicate sample) (c). The transfection was repeated every 4days and the cumulative population doublings (PDL) were examined (d). e,Knockdown of miR-34a expression partially inhibits Nutlin-3A-inducedsenescence. hTERT-immortalized human fibroblasts (hTERT/NHF) weretransfected with the antisense miR-34a or control oligonucleotide, andthen induced to senesce by treatment with 10 μM of Nutlin-3A for 72 hSummary of senescence-associated β-galactosidase (SA-β-gal) assay isshown. The data (mean±s.d.) were from three independent experiments. *,p<0.05.

FIG. 15. Knockdown of endogenous Δ133p53 induces cellular senescence.Early-passage WI-38 fibroblasts (at passage 30) were transfected withsiRNAs targeting Δ133p53 (Δ133si-1 and Δ133si-2) and a controloligonucleotide twice (at day 1 and day 4), and at day 7 were used forimmunoblot analyses (a) and examined for SA-β-gal activity (b and c),bromo-deoxyuridine (BrdU) incorporation (d) and PAI-1 (plasminogenactivator inhibitor-1) expression (e). a, siRNA-mediated repression ofΔ133p53. Expressions of full-length p53 (DO-1 antibody), Δ133p53 (MAP4antibody), p53β (TLQ40 antibody) and p21^(WAF1) were examined Theexpression levels of full-length p53 and Δ133p53 were also confirmed bythe CM1 antibody. β-actin was a loading control. H1299 expressing p53βwas the positive control for TLQ40. b, Representative pictures ofSA-β-gal staining c, Summary of SA-β-gal assay. The data (mean±s.d.)were from three independent experiments. *, p<0.01. d, BrdUincorporation assay. The number of BrdU-positive cells/the total numberof cells examined (at least 100 cells for each well) was recorded. Dataare mean±s.d. from triplicate wells. *, p<0.05. **, p<0.01. e, Thereal-time qRT-PCR assay of PAI-1. The relative expression levels ofPAI-1 mRNA are shown. Error bars represent s.d. from triplicate sample.*, p<0.05. **, p<0.01.

FIG. 16. Overexpression of p53β induces senescence and overexpression ofΔ133p53 extends replicative lifespan. Effects of retrovirallyoverexpressed p53β and Δ133p53 on cell proliferation and senescence. a,Early-passage MRC-5 and WI-38 fibroblasts (both at passage 32) wereretrovirally transduced with vector alone (open squares), full-lengthp53 (open diamonds), FLAG-tagged p53β (closed circles) and FLAG-taggedΔ133p53 (closed triangles), and used in cell proliferation assay at 8days after retroviral transduction. The cell numbers were counted dailyand the data (mean±s.d.) were from three independent experiments. b,Summary of SA-β-gal assay. The same set of cells as in (a) were examinedat 8 days after retroviral transduction. The data (mean±s.d.) were fromthree independent experiments. *, p<0.01. c, Extension of cellularreplicative lifespan by Δ133p53. The FLAG-Δ133p53 retroviral vector(open circles) or the control vector (open squares) was transduced tohuman fibroblasts at late passage (MRC-5 at passage 53 and WI-38 atpassage 51). The cumulative PDL were calculated and plotted to daysafter G418 selection. d, SA-β-gal staining of control andΔ133p53-overexpressing MRC-5 fibroblasts. The pictures at 36 dayspost-selection are shown. e, Repression of miR-34a expression byΔ133p53. RNA samples from MRC-5 (at passage 53) before transduction (day0), MRC-5 with control vector and MRC-5 overexpressing Δ133p53 (at days20, 36 and 96 post-selection) were analyzed as in FIG. 14 b. The valuebefore transduction was defined as 1.0 and the expression levels in theother samples were expressed as the relative values (mean±s.d. fromtriplicate sample). f, Telomere length and telomeric 3′ overhang inΔ133p53-overexpressing cells. Genomic DNA samples from MRC-5 withFLAG-Δ133p53 or control vector were used in the in-gel hybridizationwith ³²P-[CCCTAA]₄ (SEQ ID NO:5) probe under denatured (for telomerelength) and native (for telomeric 3′ overhang) conditions. Lane 1, MRC-5before transduction; lanes 2-3, vector control (days 4 and 35post-selection); lanes 4-6, FLAG-Δ133p53 (days 4, 35 and 96post-selection). The telomere lengths were measured as peak TRF(terminal restriction fragment) lengths. The amounts of telomeric 3′overhang were normalized with loaded DNA amounts (EtBr) and shown aspercent signals to the cells before transduction.

FIG. 17. p53 isoform expression profiles in colon carcinogenesis invivo. Elevated expression of p53β and reduced expression of Δ133p53 incolon adenomas with senescent phenotypes, but not in colon carcinomas.(a) SA-β-gal staining of non-adenoma and adenoma tissues. The results ofcase 7 are shown. The rectangular areas are enlarged in the rightpanels. Bars, 500 μm. (b) The expression levels of p53β and Δ133p53 werequantitatively examined in 9 normal colon tissues obtained fromimmediate autopsy²¹ (Table 1), 8 matched pairs of non-adenoma andadenoma tissues (Table 2) and 29 matched pairs of non-carcinoma andcarcinoma tissues (Table 3). The data (mean and s.d.) are shown in alogarithmic scale as the relative values to normal colon samples. *,p<0.05 compared with normal colon. (c) The expression levels of p53β andΔ133p53 in colon carcinomas were analyzed according to tumour stage. Thedata of normal colon and adenoma samples are same as those in (b). Theexpression levels (mean and s.d.) in adenomas, stage I (n=8), stage II(n=11) and stage III (n=10) carcinomas are shown as relative log2 valuesto normal colon (defined as 0, not shown). *, p<0.05.

FIG. 18. SA-β-gal staining in replicative senescence andoncogene-induced premature senescence. a, MRC-5 and WI-38 fibroblasts atearly passage (upper panels) and at replicative senescence (lowerpanels). b, MRC-5 and WI-38 retrovirally transduced with vector control(upper panels) and pBabe-Puro ras (H-RasV12) (Serrano et al. Cell 88,593-602 (1997)) (lower panels). Note that premature senescence by POT1knockdown was induced and confirmed by SA-13-gal staining as describedin our previous study (Yang et al. Cancer Res. 67, 11677-11686 (2007)).The dominant-negative TRF2-induced senescence was also as previouslydescribed by the present inventors (Yang et al. Mol. Cell. Biol. 25,1070-1080 (2005)) and others (van Steensel et al. Cell 92, 401-413(1998)).

FIG. 19. MAP4 specifically recognizes Δ133p53. H1299 cells (p53-null)transfected with the expression vector for wild-type (wt) p53, p53β orΔ133p53 were analyzed in immunoblot using MAP4 (left) and DO-1 (right)antibodies. MAP4 detects Δ133p53, but not wt p53 or p53β.

FIG. 20. p53 isoform switching does not occur with premature senescence.Δ133p53 and p53β expression in oncogene-induced senescence(overexpression of H-RasV12) (Serrano et al. Cell 88, 593-602 (1997))(a) and premature senescence with acute telomere dysfunction induced byshRNA knockdown of POT1 (Yang et al. Cancer Res. 67, 11677-11686 (2007))(b) or overexpression of a dominant-negative TRF2 mutant (Yang et al.Mol. Cell. Biol. 25, 1070-1080 (2005); van Steensel et al. Cell 92,401-413 (1998)) (c). Early-passage MRC-5 and WI-38 (at passage 32) wereused. H1299 cells overexpressing p53β was the positive control for p53β.β-actin was a loading control.

FIG. 21. miR-34a expression is p53-dependent. hTERT-immortalized humanfibroblasts (hTERT/NHF) (Sengupta et al. EMBO J. 22, 1210-1222 (2003))transduced with the shRNA knockdown vector targeting p53 (Brummelkampand Agami Science 296, 550-553 (2002)) (left) or treated with 10 μM ofNutlin-3a for 36 h (Kumamoto et al. Cancer Res. 68, 3193-3203 (2008))(right) were examined for miR-34a expression, as in FIG. 14 b. The data(mean±s.d. from triplicate sample) is shown as the relative expressionlevel to control cells (−).

FIG. 22. Knockdown of endogenous Δ133p53 induces cellular senescence.Early-passage MRC-5 fibroblasts (at passage 32) were transfected withthe siRNAs targeting Δ133p53 (Δ133si-1 and Δ133si-2) and a controloligonucleotide and examined in immunoblot analyses (a), SA-β-gal assay(b) and BrdU incorporation assay (c), as performed in FIG. 2. *,p<0.001.

FIG. 23. Δ133p53 knockdown does not induce apoptosis in humanfibroblasts. MRC-5 and WI-38 transfected with control, Δ133si-1 andΔ133si-2 oligonucleotides were examined for caspase-3 (top) and PARP(middle, short and long exposure) in immunoblot. RKO cells treated withdoxorubicin (DOX) were included as the positive control showingapoptosis. β-actin was a loading control (bottom). No cleaved caspase-3or PARP was observed in Δ133p53-knocked-down fibroblasts.

FIG. 24. mir-34a is not upregulated at Δ133p53 knockdown-inducedsenescence. MRC-5 and WI-38 transfected with control, Δ133si-1 andΔ133si-2 oligonucleotides were examined for miR-34a expression, as inFIG. 14 b, together with untransfected early-passage (Y) andreplicatively senescent (R.S.) cells. The data (mean±s.d. fromtriplicate sample) are shown as the relative expression levels tountransfected early-passage cells (Y, −).

FIG. 25. Retroviral overexpression of p53 isoforms in human fibroblasts.The retroviral vectors driving full-length p53, FLAG-tagged p53β andFLAG-tagged Δ133p53 were transduced to human fibroblasts at earlypassage (at passage number 30 for both strains) and the immunoblotanalyses of the overexpressed full-length p53 and p53 isoforms, MDM2 andp21^(WAF1) were performed. Protein samples were prepared from the cellsat 8 days after retroviral transduction. The anti-FLAG antibody detectedFLAG-tagged p53β and FLAG-tagged Δ133p53. The DO-12 antibody detectedfull-length p53, FLAG-tagged p53β and FLAG-tagged Δ133p53. β-actin was aloading control.

FIG. 26. p53β overexpression induces cellular senescence in humanfibroblasts with ectopically expressed telomerase. a, Effects of p53β oncell proliferation. hTERT/NHF cells (Sengupta et al. EMBO J. 22,1210-1222 (2003)) were transduced with the retroviral vector drivingFLAG-tagged p53β or control vector (a zeocin-resistant version).Cellproliferation assay was carried out as in FIG. 16 a. b, Upregulationof p21^(WAF1) by p53β overexpression in hTERT/NHF cells. c,Representative pictures of SA-β-gal staining. d, Summary of SA-β-galstaining. The data were mean±s.d. from three independent experiments. *,p<0.01.

FIG. 27. Δ133p53 overexpression extends the replicative lifespan inhuman fibroblasts. Late-passage MRC-5 (at passage 55) and WI-38 (atpassage 53) were transduced with the FLAG-Δ133p53 retroviral vector orthe control vector and examined for the cumulative PDL, as in FIG. 16 c.

FIG. 28. Immunoblot analyses of p16^(INK4A), Δ133p53 and p53β in humancolon adenomas. Eight cases of matched non-adenoma (N) and adenoma (A)tissues were examined for p16^(INK4A), Δ133p53 and p53β. β-actin was thecontrol for quantitation. The data shown in FIGS. 34 e and 4 f were fromthe quantitative analysis of these results.

FIG. 29. Increased p16^(INK4) a expression in colon adenomas. Theexpression levels of p16^(INK4A), an in vivo senescence marker, wereexamined in 9 normal colon tissues (Table 1) and 8 pairs of non-adenomaand adenoma tissues (Table 2) and quantitatively analyzed. The data(mean±s.d.) are shown as the relative values to normal colon samples. *,p<0.0001.

FIG. 30. Paired t-test analyses of W^(INK4a), Δ133p53 and p53βexpression in matched colon adenoma and non-adenoma tissues. The samedata as in FIG. 17 b and FIG. 29 from 8 pairs of non-adenoma (Non-ad)and adenoma tissues were analyzed by paired t-test. The vertical axesare the expression levels normalized with β-actin. The p-values forp16^(INK4A), Δ133p53 and p53β are 0.0004, 0.024 and 0.03, respectively,and the corresponding Bonferroni corrected p-values are 0.001, 0.07 and0.09, respectively. Case 1, aqua; case 2, blue; case 3, cyan; case 4,yellow; case 5, lavender; case 6, navy; case 7, purple; and case 8,brown.

FIG. 31. Immunoblot analyses of Δ133p53 and p53β expression in matchedcolon carcinoma and non-carcinoma tissues. Twenty-nine cases of matchedcolon carcinoma (T) and non-carcinoma (N) tissues (Table 3) wereexamined for Δ133p53 and p53β. β-actin was the control fornormalization. Each of the six SDS-PAGE gels included 5 pairs ofcarcinoma/non-carcinoma tissues, as well as the same set of normalcolon, non-adenoma and adenoma samples, which allowed quantitativecomparisons among different blots and different histopathological types,as in FIGS. 17 b and c. One case (12375) was duplicated. The data shownin FIG. 17 b (Non-ca and Ca), 4c (Carcinoma, stage I, II and III) andFIG. 32 were from the quantitative analysis of these results.

FIG. 32. Paired t-test analyses of Δ133p53 and p53β expression in p53‘wild-type’ versus ‘mutant’ cases of colon carcinomas. Twenty-eightcases of colon carcinomas were divided into two subgroups assumedly with‘wild-type’ or ‘mutant’ p53, based on the immunohistochemical stainingof p53 and MDM2 (Costa et al., The Journal of pathology 176, 45-53(1995); Nenutil et al., The Journal of pathology 207, 251-259 (2005)).In each subgroup, the expression levels of Δ133p53 (a) and p53β (b) werecompared between non-carcinoma (Non-ca) and carcinoma tissues by pairedt-test. The vertical axes are the expression levels normalized withβ-actin. The p-values are in the parentheses. The p53 ‘wild-type’carcinomas, but not “mutant” carcinomas, expressed significantly higherlevels of Δ133p53. p53β was significantly less abundant in carcinomatissues in both subgroups because of the marked increase innon-carcinoma tissues (FIG. 17 b). The actual values in each of the 28cases are shown in Table 4.

FIG. 33. IL-8 and IL-8R expression in colon adenoma and carcinomatissues. The mRNA expression levels of IL-8 (upper panel) and IL-8R(lower panel) were examined by qRT-PCR in 8 matched pairs of non-adenomaand adenoma tissues (Table 2) and 29 matched pairs of non-carcinoma andcarcinoma tissues (Table 3). The expression levels (mean and s.d.) innon-carcinoma, adenoma and carcinoma samples are shown as relative log2values to non-adenoma (defined as 0). *, p<0.05 compared withnon-adenoma or non-carcinoma. **, p<0.001 compared with non-adenoma ornon-carcinoma.

FIG. 34. p53 isoform switching in vivo. a-c, Increased p53β anddecreased Δ133p53 expression during CD8⁺ T lymphocyte senescence invivo. a, CD8⁺ T lymphocytes were purified from blood samples freshlyisolated from healthy donors of age 50 years old, and sorted by flowcytometry using anti-CD28 and anti-CD57 antibodies. The result of50-year-old male is shown. b, Representative immnunoblot of p53β andΔ133p53. The results of 65-year-old male are shown. HP1-y was examinedas a senescence marker. β-actin was a loading control for quantitation.c, The expression levels of p53β and Δ133p53 in each of the quadrantswere quantitated in immunoblot analyses and shown as the relative valuesto the CD28⁻CD57⁺ quadrant (p53β) or CD28⁺CD57⁻ quadrant (Δ133p53). Thedata (mean±s.d.) were from three donors (60-year-old female, 65-year-oldmale and 50-year-old male). The p-values from ANOVA trend analysis areshown. d-f, Elevated expression of p53β and reduced expression ofΔ133p53 in colon adenomas with senescent phenotypes. d, SA-β-galstaining of non-adenoma and adenoma tissues. The results of case 7 areshown. The rectangular areas are enlarged in the right panels. Bars, 500μm. e, The expression levels of p53β and Δ133p53, as well as asenescence marker p16^(INK4A) were examined in 9 normal colon tissuesobtained from immediate autopsy (Table 1) and 8 matched pairs ofnon-adenoma and adenoma tissues surgically resected (Table 2) andquantitatively analyzed. The data (mean±s.d.) are shown in a logarithmicscale as the relative values to normal colon samples. *, p<0.05. **,p<0.0005. ***, p<0.00005. f, The same data as in (e) from 8 matchedpairs of non-adenoma (Non-ad) and adenoma tissues were analyzed bypaired t-test. The vertical axes are the expression levels normalizedwith β-actin. The p-values for p16^(INK4A) Δ133p53 and p53β are 0.0004,0.024 and 0.03, respectively, and the corresponding Bonferroni correctedp-values are 0.001, 0.07 and 0.09, respectively. Case 1, aqua; case 2,blue; case 3, cyan; case 4, yellow; case 5, lavender; case 6, navy; case7, purple; and case 8, brown.

FIG. 35. mRNA expression analysis of p53 isoforms in human fibroblasts.The same sets of cells as in FIG. 14 a were analyzed by RT-PCR. Theprimers to amplify wt p53 were: 5′-CTC ACC ATC ATC ACA CTG GAA-3′ (SEQID NO:6) and 5′-TCA TTC AGC TCT CGG AAC ATC-3′ (SEQ ID NO:7). Theprimers specifically detecting the alternative splicing for p53β were:5′-CTT TGA GGT GCG TGT TTG TGC-3′ (SEQ ID NO:8) and 5′-TTG AAA GCT GGTCTG GTC CTG A-3′ (SEQ ID NO:9). The primers specifically amplifyingΔ133p53 mRNA transcribed from the promoter in intron 4 were: 5′-TGG GTTGCA GGA GGT GCT TAC-3′ (SEQ ID NO:10) and 5′-CCA CTC GGA TAA GAT GCT GAGG-3′ (SEQ ID NO:11). The lower bands correspond to the reported Δ133p53sequences (GenBank DQ186650). The upper bands are from mRNA with intron5 unspliced. GAPDH was amplified as a control as previously described(Horikawa and Barrett Mol. Carcinog. 22, 65-72 (1998)).

FIG. 36. FACS (Fluorescence-activated cell sorting) of human CD8⁺ Tlymphocytes. a, Summary of the sorted fractions from three donors. b,The purity of sorted fractions was checked by FACS reanalysis. Theresult of 50-year-old male is shown. c, Immunoblot analysis of thesorted fractions for HP1-y as a senescence marker (Collado et al. Nature436, 642 (2005); Narita et al. Cell 113, 703-716 (2003); Zhang et al. J.Cell Science 120, 1572-1583 (2007)). The expression levels of HP1-γ werequantitated and expressed as the relative values to CD28⁺CD57⁻ fraction.The data (mean±s.d.) were from three donors. The difference betweenCD28⁺CD57⁻ and CD28⁻CD57⁺ fractions is statistically significant (p<0.05).

FIG. 37. Δ133p53 and p53 β expression in human CD8⁺ T lymphocytes.Immunoblot analysis as shown in FIG. 14 b. a, 60-year-old female. b,50-year-old male.

FIG. 38. Δ133p53 is not subject to proteasomal degradation.Early-passage (Y) and replicatively senescent (S) MRC-5 and WI-38 (thesame set of cells as in FIG. 14 a) were maintained in the presence (+)or absence (−) of 15 μM of the proteasome inhibitor MG-132 for 8 hrs andexamined in immunoblot.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The finite division potential of normal human cells leads to cellularsenescence, which functions as a barrier to human cell transformationand carcinogenesis (Collado, M., et al., Cell 130:223 (2007)). Theinduction and prevention of cellular senescence in human cells involvethe regulation of the specific chromosome end structure, telomeres(Verdun, R. E. et al., Nature 447:924 (2007)). The tumor suppressorprotein p53 plays a central role in sensing and signaling a variety ofintrinsic stresses (e.g., telomere dysfunction) and environmental cuesthat induce cellular senescence (Collado, M. , et al., Cell 130:223(2007); Herbig, U. et al., Mol. Cell 14:501 (2004)). p53 and Arf canalso cooperate to have anti-oxidative and anti-aging activities (Matheu,A. et al., Nature 448:375 (2007)). Many of the mutant p53 proteinsobserved in human cancers inhibit the tumor suppressive functions offull-length, wild-type p53 (wt p53) in a dominant-negative manner(Rozan, L. M. et al., Cell Death Differ. 14:3 (2007)). It is suggestedthat some p53 mutants also gain a tumor-promoting function independentof the inhibition of wt p53 (Rozan, L. M. et al., Cell Death Differ.14:3 (2007); Kastan, M. B. et al., Nat. Cell Biol. 9:489 (2007)). Thehuman p53 gene encodes, in addition to wt p53, several N-terminally,internally and C-terminally truncated isoforms due to alternativepromoter usage and RNA splicing (Chan, W. M. et al., Cancer Res. 67:1959(2007), Bourdon, J. C. et al., Genes Dev. 19:2122 (2005)). A plausiblehypothesis is that these p53 isoforms cooperate or compete with wt p53to modulate the p53′s multiple functions. To test this hypothesis, weexamine here the roles of two major isoforms, p53β (lacking theC-terminal oligomerization domain due to an alternative splicing) andΔ133p53 (transcribed from the alternative promoter in intron 4 andlacking the N-terminal transactivation and proline-rich domains)(Bourdon, J. C. et al., Genes Dev. 19:2122 (2005)), in the regulation ofcellular senescence and their functional interplay with wt p53. Our dataprovide novel insights into the p53 regulation of cellular replicativelifespan.

II. p53 Proteins

p53 is a protein of apparent molecular 53 kDa on SDS PAGE that functionsas a transcription factor that, among other functions, regulates thecell cycle and functions as a tumor suppressor. p53 has been describedas “the guardian of the genome”, referring to its role in providingstability by preventing genome mutation. Among p53′s anti-canceractivities include: activation of DNA repair proteins when DNA hassustained damage; cell cycle arrest at the G1/S regulation point when acell has sustained DNA damage, thus allowing DNA repair proteins time tofix the damage before allowing continuation of the cell cycle; and theinitiation of apoptosis or the programmed cell death, if the DNA damageproves to be irreparable.

Accordingly, p53 can induce growth arrest, apoptosis, and cellsenescence. In normal cells, p53 is generally held in an inactive form,bound to the protein MDM2 (HDM2 in humans), which prevents p53 activityand promotes p53 degradation by acting as a ubiquitin ligase. Active p53is induced in response to various cancer-causing agents such as UVradiation, oncogenes, and some DNA-damaging drugs. DNA damage is sensedby ‘checkpoints’ in a cell's cycle, and causes proteins such as ATM,CHK1 and CHK2 to phosphorylate p53 at sites that are close to or withinthe MDM2-binding region and p300-binding region of the protein.Oncogenes also stimulate p53 activation, mediated by the protein p14ARF.Some oncogenes can also stimulate the transcription of proteins whichbind to MDM2 and inhibit its activity. Once activated, p53 activatesexpression of several genes including one encoding for p21, a cell cycleinhibitor. p21 binds to G1-S-phase and S-phase cyclin CDK complexesinhibiting their activity. See, e.g., Mills, Genes & Development, 19:2091-2099 (2005) for a review.

Other isoforms or variants of p53 have been identified (see Bourdon,Brit. J. Cancer, 97: 277-282 (2007)). For example, two isoforms of p53,p63 and p73, which are encoded by distinct genes, have been identified(Kaghad et al., Cell 90: 809-819 (1997); and Yang et al. Mol. Cell(1998)). Human p53 isoforms may also arise due to alternative promoterusage and alternative splicing. Alternative promoter usage, for example,can give rise to the expression of an N-terminally truncated p53 proteininitiated at codon 133 (Δ133p53). Adding to the complexity of p53isoforms is the alternative splicing of intron 9 of the p53 gene toprovide the isoforms p53β and p53γ. Combined with alternative promoterusage, this gives rise to the p53 isoforms: p53, p53β, p53γ, Δ133p53,Δ133p53β, and Δ133p53γ. The use of an alternative promoter in intron 2gives rise to the additional isoforms, Δ40p53, Δ40p53β, and Δ40p53γ.While the presence of these multiple p53 isoforms has been established,the biological function of these isoforms remains obscure. The presentinvention invention is based in part on an elucidation of the role fortwo of these isoforms, Δ133p53 and p53 β, in the opposing functions ofcell senescence and cell proliferation.

III. Definitions

The term “p53” refers generally to a protein of apparent molecularweight of 55 kDa on SDS PAGE that functions as a tumor suppressor asdescribed herein. The protein and nucleic sequences of the p53 proteinfrom a variety of organisms from humans to Drosophila are known and areavailable in public databases, such as in accession numbers,NM_(—)000546, NP_(—)000537, NM_(—)011640, and NP_(—)035770, for thehuman and mouse sequences.

The term “Δ133p53” refers generally to the isoform of p53 that arisesfrom initiation of transcription of the p53 gene from codon 133, whichresults in an N-terminally truncated p53 protein. This isoform comprisesthe following p53 protein domains: the majority of the DNA bindingdomain, the NLS, and the C-terminal sequence DQTSFQKENC (SEQ ID NO:12)(see Bourdon, Brit. J. Cancer, 97: 277-282 (2007)).

The term “p53 β” refers generally to the isoform of p53 that arises fromalternative splicing of intron 9 to provide a p53 isoform comprising thefollowing p53 protein domains: TAD1, TAD2, prD, the DNA binding domain,the NLS, and the C-terminal sequence DQTSFQKENC (SEQ ID NO:12) (seeBourdon, Brit. J. Cancer, 97: 277-282 (2007)).

The term “cell senescence” refers generally to the phenomenon wherenormal diploid differentiated cells lose the ability to divide afterundergoing a finite number of cell divisions characteristic of aparticular type of cell.

The term “replicative lifespan” refers generally to the finite number ofcell divisions undergone by a particular cell type before undergoingcell senescence and losing the ability to further divide.

The term “extending replicative lifespan” refers generally to thecontinuation of cell division in a normal diploid cell beyond the finitenumber of cell divisions at which cell senescence would occur.

The term “siRNA” refers to a nucleic acid that forms a double strandedRNA, which double stranded RNA has the ability to reduce or inhibitexpression of a gene or target gene when the siRNA expressed in the samecell as the gene or target gene. “siRNA” thus refers to the doublestranded RNA formed by the complementary strands. The complementaryportions of the siRNA that hybridize to form the double strandedmolecule typically have substantial or complete identity. In oneembodiment, an siRNA refers to a nucleic acid that has substantial orcomplete identity to a target gene and forms a double stranded siRNA.The sequence of the siRNA can correspond to the full length target gene,or a subsequence thereof Typically, the siRNA is at least about 15-50nucleotides in length (e.g., each complementary sequence of the doublestranded siRNA is 15-50 nucleotides in length, and the double strandedsiRNA is about 15-50 base pairs in length, preferable about preferablyabout 20-30 base nucleotides, preferably about 20-25 nucleotides inlength, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotidesin length.

The term “shRNA” refers generally to an siRNA that is introduced into acell as part of a larger DNA construct. Typically, such constructs allowstable expression of the siRNA in cells after introduction, e.g., byintegration of the construct into the host genome.

An “antisense” oligonucleotide or polynucleotide is a nucleotidesequence that is substantially complementary to a target polynucleotideor a portion thereof and has the ability to specifically hybridize tothe target polynucleotide.

Ribozymes are enzymatic RNA molecules capable of catalyzing specificcleavage of RNA. The composition of ribozyme molecules preferablyincludes one or more sequences complementary to a target mRNA, and thewell known catalytic sequence responsible for mRNA cleavage or afunctionally equivalent sequence (see, e.g., U.S. Pat. No. 5,093,246,which is incorporated herein by reference in its entirety). Ribozymemolecules designed to catalytically cleave target mRNA transcripts canalso be used to prevent translation of subject target mRNAs.

The term “promoting” as used, for example in the context of “promotingsenescence,” refers generally to conditions or agents which increase,induce, open, activate, facilitate, enhance activation, sensitize,agonize, or up regulate cell senescence.

The phrase “functional effects” in the context of assays for testingcompounds that modulate a protein of the invention includes thedetermination of a parameter that is indirectly or directly under theinfluence of a protein of the invention, e.g., a chemical or phenotypiceffect such as altered transcriptional activity of p53 isoforms and thedownstream effects of such proteins on cellular metabolism andproliferation or growth. A functional effect therefore includestranscriptional activation or repression, the ability of cells toproliferate or undergo apoptosis, whether and at what point cellsundergo senescence, among others. “Functional effects” include in vitro,in vivo, and ex vivo activities.

By “determining the functional effect” is meant assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of a p53 isoform of the invention, e.g., measuringphysical and chemical or phenotypic effects. Such functional effects canbe measured by any means known to those skilled in the art, e.g.,changes in spectroscopic characteristics (e.g., fluorescence,absorbance, refractive index); hydrodynamic (e.g., shape),chromatographic; or solubility properties for the protein; ligandbinding assays, e.g., binding to antibodies; measuring inducible markersor transcriptional activation of the marker; measuring changes inenzymatic activity; the ability to increase or decrease cellularproliferation, senescence, apoptosis, cell cycle arrest, measuringchanges in cell surface markers. The functional effects can be evaluatedby many means known to those skilled in the art, e.g., microscopy forquantitative or qualitative measures of alterations in morphologicalfeatures, measurement of changes in RNA or protein levels for othergenes expressed in a cell, measurement of RNA stability, identificationof downstream or reporter gene expression (CAT, luciferase, β-gal, GFPand the like), e.g., via chemiluminescence, fluorescence, colorimetricreactions, antibody binding, inducible markers, etc.

“Inhibitors,” “activators,” and “modulators” of the proteins of theinvention are used to refer to activating, inhibitory, or modulatingmolecules identified using in vitro and in vivo assays of p53 isoforms.Inhibitors are compounds that, e.g., bind to, partially or totally blockactivity, decrease, prevent, delay activation, inactivate, desensitize,or down regulate the activity or expression of p53 isoforms.“Activators” are compounds that increase, open, activate, facilitate,enhance activation, sensitize, agonize, or up regulate activity of p53isoforms, e.g., agonists. Inhibitors, activators, or modulators alsoinclude genetically modified versions of p53 isoforms, e.g., versionswith altered activity, as well as naturally occurring and syntheticligands, antagonists, agonists, antibodies, peptides, cyclic peptides,nucleic acids, antisense molecules, ribozymes, RNAi molecules, smallorganic molecules and the like. Such assays for inhibitors andactivators include, e.g., expressing p53 isoforms in vitro, in cells, orcell extracts, applying putative modulator compounds, and thendetermining the functional effects on activity, as described above.

Samples or assays comprising p53 isoforms that are treated with apotential activator, inhibitor, or modulator are compared to controlsamples without the inhibitor, activator, or modulator to examine theextent of inhibition. Control samples (untreated with inhibitors) areassigned a relative protein activity value of 100%. Inhibition of p53isoforms is achieved when the activity value relative to the control isabout 80%, preferably 50%, more preferably 25-0%. Activation of p53isoforms is achieved when the activity value relative to the control(untreated with activators) is 110%, more preferably 150%, morepreferably 200-500% (i.e., two to five fold higher relative to thecontrol), more preferably 1000-3000% higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, peptide,circular peptide, lipid, fatty acid, siRNA, polynucleotide,oligonucleotide, etc., to be tested for the capacity to directly orindirectly modulate p53 isoforms. The test compound can be in the formof a library of test compounds, such as a combinatorial or randomizedlibrary that provides a sufficient range of diversity. Test compoundsare optionally linked to a fusion partner, e.g., targeting compounds,rescue compounds, dimerization compounds, stabilizing compounds,addressable compounds, and other functional moieties. Conventionally,new chemical entities with useful properties are generated byidentifying a test compound (called a “lead compound”) with somedesirable property or activity, e g., inhibiting activity, creatingvariants of the lead compound, and evaluating the property and activityof those variant compounds. Often, high throughput screening (HTS)methods are employed for such an analysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

IV. Nucleic Acids and Proteins of the Invention A. General RecombinantDNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Generally, the nomenclature and the laboratory procedures inrecombinant DNA technology described below are those well known andcommonly employed in the art. Standard techniques are used for cloning,DNA and RNA isolation, amplification and purification. Generallyenzymatic reactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like are performed according to the manufacturer'sspecifications. Basic texts disclosing the general methods of use inthis invention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Methods for Isolating Nucleotide Sequences Encoding Δ133p53 or p53β

In general, the nucleic acid sequences encoding Δ133p53 or p53β andrelated nucleic acid sequence homologues can be cloned from cDNAlibraries or isolated using amplification techniques witholigonucleotide primers. Nucleic acids encoding Δ133p53 or p53β can alsobe isolated from expression libraries using antibodies as probes.

Advantageously, the cloning of Δ133p53 or p53β or other p53 isoforms canemploy the use of synthetic oligonucleotide primers and amplification ofan RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCRProtocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of Δ133p53or p53β directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. Degenerate oligonucleotides can be designed to amplifyΔ133p53 or p53β homologues for other species using known sequences.Restriction endonuclease sites can be incorporated into the primers.Genes amplified by the PCR reaction can be purified from agarose gelsand cloned into an appropriate vector.

The nucleic acids encoding Δ133p53 or p53β or other p53 isoforms aretypically cloned into intermediate vectors before transformation intoprokaryotic or eukaryotic cells for replication and/or expression. Theseintermediate vectors are typically prokaryote vectors, e.g., plasmids,or shuttle vectors. The isolated nucleic acids encoding Δ133p53 or p53βor other p53 isoforms comprise nucleic acid sequences these proteins andsubsequences, interspecies homologues, alleles and polymorphic variantsthereof.

C. Expression of Δ133p53 or p53β in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding Δ133p53 or p53β, one typically subclones Δ133p53 or p53βnucleic acids into an expression vector that contains a strong promoterto direct transcription, a transcription/translation terminator, and iffor a nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing Δ133p53 or p53 β proteinsare available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva etal., Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983).Kits for such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of Δ133p53 or p53βencoding nucleic acid in host cells. A typical expression cassette thuscontains a promoter operably linked to the nucleic acid sequenceencoding Δ133p53 or p53 β proteins and signals required for efficientpolyadenylation of the transcript, ribosome binding sites, andtranslation termination. Additional elements of the construct mayinclude enhancers and, if genomic DNA is used as the structural gene,introns with functional splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

Many conventional vectors for transport of genetic information into acell may be used for expression in eukaryotic or prokaryotic cells maybe used. Standard bacterial expression vectors include plasmids such aspBR322 based plasmids, pSKF, pET23D, and fusion expression systems suchas GST and LacZ. Epitope tags can also be added to recombinant proteinsto provide convenient methods of isolation and detection, e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Standard transfection methods may be used to introduce the nucleic acidconstructs of the invention into bacterial, mammalian, yeast or insectcell lines. Transformation of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison, J.Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983). These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra).

An advantageous expression system involves the use of retroviralexpression vectors to express the constructs of the invention. After thecloning of a suitable nucleic acid encoding Δ133p53 or p53β or aninhibitory nucleic acid into an appropriate retroviral vector, thenucleic acid constructs are transfected into an appropriate retroviralpackaging cell such as PE 501, BOSC, ψCRE, GP+E-86, PA317, ψCRIP,GP+envAm12, and Phoenix, among others, depending on the cell type to beultimately infected with the resulting retrovirus (see, e.g.,Recombinant Gene Expression Protocols in Methods in Molecular Biol.,vol. 62, ed. R. Tuan, Humana Press (1997)).

V. Inhibition of p53 Isoforms Using Nucleic Acids

Inhibitory nucleic acids to Δ133p53 or p53β, such as siRNA, shRNA,ribozymes, or antisense molecules, can be synthesized and introducedinto cells using methods known in the art. Molecules can be synthesizedchemically or enzymatically in vitro (Micura, Agnes Chem. Int. Ed. Emgl.41: 2265-9 (2002); Paddison et al., Proc. Natl. Acad. Sci. USA, 99:1443-8 2002) or endogenously expressed inside the cells in the form ofshRNAs (Yu et al., Proc. Natl. Acad. Sci. USA, 99: 6047-52 (2002);McManus et al., RNA 8, 842-50 (2002)). Plasmid-based expression systemsusing RNA polymerase III U6 or H1, or RNA polymerase II U1, smallnuclear RNA promoters, have been used for endogenous expression ofshRNAs (Brummelkamp et al., Science, 296: 550-3 (2002); Sui et al.,Proc. Natl. Acad. Sci. USA, 99: 5515-20 (2002); Novarino et al., J.Neurosci., 24: 5322-30 (2004)). Synthetic siRNAs can be delivered byelectroporation or by using lipophilic agents (McManus et al., RNA 8,842-50 (2002); Kishida et al., J. Gene Med., 6: 105-10 (2004)).Alternatively, plasmid systems can be used to stably express smallhairpin RNAs for the suppression of target genes (Dykxhoorn et al., Nat.Rev. Mol. Biol., 4: 457-67 (2003)). Various viral delivery systems havebeen developed to deliver shRNA-expressing cassettes into cells that aredifficult to transfect (Brummelkamp et al., Cancer Cell, 2: 243-7(2002); Rubinson et al., Nat. Genet., 33: 401-6 2003). Furthermore,siRNAs can also be delivered into live animals. (Hasuwa et al., FEBSLett., 532, 227-30 (2002); Carmell et al., Nat. Struct. Biol., 10: 91-2(2003); Kobayashi et al., J. Pharmacol. Exp. Ther., 308: 688-93 (2004)).

Methods for the design of siRNA or shRNA target sequences have beendescribed in the art. Among the factors to be considered include: siRNAtarget sequences should be specific to the gene of interest and have˜20-50% GC content (Henshel et al., Nucl. Acids Res., 32: 113-20 (2004);G/C at the 5′ end of the sense strand; A/U at the 5′ end of theantisense strand; at least 5 A/U residues in the first 7 bases of the 5′terminal of the antisense strand; and no runs of more than 9 G/Cresidues (Ui-Tei et al., Nucl. Acids Res., 3: 936-48 (2004)).Additionally, primer design rules specific to the RNA polymerase willapply. For example, for RNA polymerase III, the polymerase thattranscribes from the U6 promoter, the preferred target sequence is5′-GN18-3′. Runs of 4 or more Ts (or As on the other strand) will serveas terminator sequences for RNA polymerase III and should be avoided. Inaddition, regions with a run of any single base should be avoided(Czauderna et al., Nucl. Acids Res., 31: 2705-16 (2003)). It has alsobeen generally recommended that the mRNA target site be at least 50-200bases downstream of the start codon (Sui et al., Proc. Natl. Acad. Sci.USA, 99: 5515-20 (2002); Elbashir et al., Methods, 26: 199-213 (2002);Duxbury and Whang, J. Surg. Res., 117: 339-44 (2004) to avoid regions inwhich regulatory proteins might bind. Additionally, a number of computerprograms are available to aid in the design of suitable siRNA and shRNAsfor use in the practice of this invention.

Ribozymes that cleave mRNA at site-specific recognition sequences can beused to destroy target mRNAs, particularly through the use of hammerheadribozymes. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. Preferably, the target mRNA has the following sequence of twobases: 5′-UG-3′. The construction and production of hammerhead ribozymesis well known in the art.

Gene targeting ribozymes necessarily contain a hybridizing regioncomplementary to two regions, each of at least 5 and preferably each 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguousnucleotides in length of a target mRNA. In addition, ribozymes possesshighly specific endoribonuclease activity, which autocatalyticallycleaves the target sense mRNA.

With regard to antisense, siRNA or ribozyme oligonucleotides,phosphorothioate oligonucleotides can be used. Modifications of thephosphodiester linkage as well as of the heterocycle or the sugar mayprovide an increase in efficiency. Phophorothioate is used to modify thephosphodiester linkage. An N3′-P5′ phosphoramidate linkage has beendescribed as stabilizing oligonucleotides to nucleases and increasingthe binding to RNA. Peptide nucleic acid (PNA) linkage is a completereplacement of the ribose and phosphodiester backbone and is stable tonucleases, increases the binding affinity to RNA, and does not allowcleavage by RNAse H. Its basic structure is also amenable tomodifications that may allow its optimization as an antisense component.With respect to modifications of the heterocycle, certain heterocyclemodifications have proven to augment antisense effects withoutinterfering with RNAse H activity. An example of such modification isC-5 thiazole modification. Finally, modification of the sugar may alsobe considered. 2′-O-propyl and 2′-methoxyethoxy ribose modificationsstabilize oligonucleotides to nucleases in cell culture and in vivo.

Inhibitory oligonucleotides can be delivered to a cell by directtransfection or transfection and expression via an expression vector.Appropriate expression vectors include mammalian expression vectors andviral vectors, into which has been cloned an inhibitory oligonucleotidewith the appropriate regulatory sequences including a promoter to resultin expression of the antisense RNA in a host cell. Suitable promoterscan be constitutive or development-specific promoters. Transfectiondelivery can be achieved by liposomal transfection reagents, known inthe art (e.g., Xtreme transfection reagent, Roche, Alameda, CA;Lipofectamine formulations, Invitrogen, Carlsbad, Calif.). Deliverymediated by cationic liposomes, by retroviral vectors and directdelivery are efficient. Another possible delivery mode is targetingusing antibody to cell surface markers for the target cells.

For transfection, a composition comprising one or more nucleic acidmolecules (within or without vectors) can comprise a delivery vehicle,including liposomes, for administration to a subject, carriers anddiluents and their salts, and/or can be present in pharmaceuticallyacceptable formulations. Methods for the delivery of nucleic acidmolecules are described, for example, in Gilmore, et al., Curr DrugDelivery (2006) 3:147-5 and Patil, et al., AAPS Journal (2005)7:E61-E77, each of which are incorporated herein by reference. Deliveryof siRNA molecules is also described in several U.S. PatentPublications, including for example, 2006/0019912; 2006/0014289;2005/0239687; 2005/0222064; and 2004/0204377, the disclosures of each ofwhich are hereby incorporated herein by reference. Nucleic acidmolecules can be administered to cells by a variety of methods known tothose of skill in the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, by electroporation, or byincorporation into other vehicles, including biodegradable polymers,hydrogels, cyclodextrins (see, for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and U.S. Patent Application PublicationNo. 2002/130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives.

Examples of liposomal transfection reagents of use with this inventioninclude, for example: CellFectin, 1:1.5 (M/M) liposome formulation ofthe cationic lipid N, NI, NII, NIII-tetramethyl-N, NI, NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE)(GIBCO BRL); Cytofectin GSV, 2:1 (M/M) liposome formulation of acationic lipid and DOPE (Glen Research); DOTAP(N-[1-(2,3-dioleoyloxy)-N, N, N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); Lipofectamine, 3:1 (M/M) liposome formulation ofthe polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL); and(5) siPORT (Ambion); HiPerfect (Qiagen); X-treme GENE (Roche);RNAicarrier (Epoch Biolabs) and TransPass (New England Biolabs).

In some embodiments, antisense, siRNA, or ribozyme sequences aredelivered into the cell via a mammalian expression vector. For example,mammalian expression vectors suitable for siRNA expression arecommercially available, for example, from Ambion (e.g., pSilencervectors), Austin, Tex.; Promega (e.g., GeneClip, siSTRIKE, SiLentGene),Madison, Wisc.; Invitrogen, Carlsbad, Calif.; InvivoGen, San Diego,Calif.; and Imgenex, San Diego, Calif. Typically, expression vectors fortranscribing siRNA molecules will have a U6 promoter.

In some embodiments, antisense, siRNA, or ribozyme sequences aredelivered into cells via a viral expression vector. Viral vectorssuitable for delivering such molecules to cells include adenoviralvectors, adeno-associated vectors, and retroviral vectors (includinglentiviral vectors). For example, viral vectors developed for deliveringand expressing siRNA oligonucleotides are commercially available from,for example, GeneDetect, Bradenton, Fla.; Ambion, Austin, Tex.;Invitrogen, Carlsbad, Calif.; Open BioSystems, Huntsville, Al.; andImgenex, San Diego, Calif.

VI. Assays of Cell Senescence, Cell Proliferation, and Apoptosis

Any of a number of known methods for the determination and measurementof cell senescence, cell proliferation, and apoptosis may be used in thepractice of this invention. Direct measurements of cell proliferationinclude direct counting of cells using, e.g., a hematocytometer,measurement of the incorporation of labeled DNA precursors such as³H-thymidine and BrdU, or through the measurement of cell markers thatare expressed in proliferating cells, such PCNA, or by measurement of amarker for cellular metabolism such as MTT (see, e.g., Hughes, D., Cellproliferation and apoptosis, Taylor & Francis Ltd, UK (2003). Othermethods such as soft agar growth or colony formation in suspension,contact inhibition and density limitation of growth, or growth factor orserum dependence of growth, among others, may be used to assess cellgrowth, especially of cancer cells as compared to normal cells (see,e.g., Freshney, Culture of Animal Cells a Manual of Basic Technique, 3rded., Wiley-Liss, New York (1994)).

A number of markers for cell senescence may be used to monitor thisprocess in the practice of this invention. The most common of thesemarkers is senescence-associated-β-galactoside (Dimri, G. P. et al.,Proc. Natl. Acad. Sci. USA 92:9363 (1995)), although others such, as thedirect measurement of telomere length by in situ hybridization, andage-dependent cellular accumulation of lipofucin in cells (Coates, J.Pathol., 196: 371-3 (2002)), are also known.

Typical assays used to detect and measure apoptosis include microscopicexamination of cellular morphology, TUNEL assays for DNA fragmentation ,caspase activity assays, annexin-V externalization assays, and DNAladdering assays, among others (see, e.g., Hughes, D., Cellproliferation and apoptosis, Taylor & Francis Ltd, UK (2003)).

VII. Methods to Identify Modulators

A variety of methods may be used to identify compounds that modulate p53isoforms. Typically, an assay that provides a readily measured parameteris adapted to be performed in the wells of multi-well plates in order tofacilitate the screening of members of a library of test compounds asdescribed herein. Thus, in one embodiment, an appropriate number ofcells or other suitable preparation can be plated into the cells of amulti-well plate, and the effect of a test compound on a p53 isoform canbe determined

The compounds to be tested can be any small chemical compound, or amacromolecule, such as a protein, sugar, nucleic acid or lipid.Typically, test compounds will be small chemical molecules and peptides.Essentially any chemical compound can be used as a test compound in thisaspect of the invention, although most often compounds that can bedissolved in aqueous or organic (especially DMSO-based) solutions areused. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods are usedwhich involve providing a combinatorial chemical or peptide librarycontaining a large number of potential therapeutic compounds. Such“combinatorial chemical libraries” or “ligand libraries” are thenscreened in one or more assays, as described herein, to identify thoselibrary members (particular chemical species or subclasses) that displaya desired characteristic activity. In this instance, such compounds arescreened for their ability to reduce or increase the function orexpression of the p53 isoforms of the invention.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries are wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991)and Houghton et al., Nature, 354:84-88 (1991)). Other chemistries forgenerating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., PNASUSA, 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J.Amer. Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimetics withglucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.,114:9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)),oligocarbamates (Cho et al., Science, 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/U.S. 96/10287), carbohydrate libraries (see, e.g., Lianget al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853),small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,Jan 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. Nos. 5,506,337; benzodiazepines, 5,288,514, and thelike).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 96 modulators. If 1536 well plates are used, thena single plate can easily assay from about 100- about 1500 differentcompounds. It is possible to assay many plates per day; assay screensfor up to about 6,000, 20,000, 50,000, or 100,000 or more differentcompounds is possible using the integrated systems of the invention.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Methods and Materials Cells

CC1, a human choriocarcinoma cell line expressing Δ133p53 due to thegenomic rearrangement deleting the exons 2, 3 and 4 (Horikawa, I. etal., Hacm. Mol. Genet. 4:313 (1995)), was a gift from Dr. MitsuoOshimura (Tottori University, Japan). Fibroblasts from Li-Fraumenisyndrome patients (MDAH041, MDAH087 and MDAH172) (Bischoff, F. Z. etal., Cancer Res. 50:7979 (1990)) were kindly provided by Dr. MichaelTainsky (Case Western Reserve University, Cleveland, Ohio). Normal humanfibroblast strains (MRC-5 and

WI-38), H1299 and 293T were obtained from American Type CultureCollection (Manassas, VA). hTERT/NHF, an hTERT (human telomerase reversetranscriptase)-immortalized human fibroblast cell line, was previouslydescribed (Sengupta, S. et al., EMBO J. 22:1210 (2003)).

Plasmid Constructs

To generate the retroviral expression vectors of human p53 isoforms,full-length p53, FLAG-tagged p53β and FLAG-tagged Δ133p53 werePCR-amplified using pSVrp53, pSVp53β and pSVDNp53 (Bourdon, J. C. etal., Genes Dev. 19:2122 (2005)), respectively, as the templates, andthen inserted into Not I and Eco RI sites of pQCXIN vector (BDBiosciences). A retroviral shRNA construct for p53 knockdown, targetingnucleotide positions 1026 to 1044 in NM_(—)000546 (Brummelkamp, T. R. etal., Science 296:550 (2002)), was derived from pSUPERretro vectorcarrying a ptuomycin-resistant gene (Oligoengine, Seattle, Wash.). Togenerate a retroviral vector driving the dominant-negative mutant ofSiah-1A (FLAG-Siah1-ΔRING), the human Siah-1A cDNA fragment (nucleotidepositions 325 to 966 in NM_(—)003031.3) was PCR-amplified using a 5′primer with FLAG tag sequence and cloned into pBABE-puro. The resultingconstruct drives an N-terminally deleted Siah-1A protein (residues 70 to282) missing the RING finger domain (Hu, G. et al., Mol. Cell. Biol.19:724 (1999)). For a retroviral vector driving FLAG-tagged Siah1-A6 (astable form of Siah-1A, consisting of residues 6 to 282) (Tanikawa, J.et al., J. Biol. Chem. 279:55393 (2004)), the human Siah-1A cDNAfragment (nucleotide positions 133 to 966 in NM_(—)003031.3) wasamplified and processed in the same way. These constructs were verifiedby DNA sequencing. The retroviral construct pLPC-Myc-TRF2 was a giftfrom Dr. Titia de Lange (Rockefeller University, N.Y.).

Retroviral Vector Production and Transduction

The retroviral constructs were transfected into Phoenix packaging cells(Orbigen, Inc.) using Lipofectamin 2000 (Invitrogen). Vectorsupernatants were collected 48 h after transfection and used to infectcells in the presence of 8 μg/ml polybrene (Sigma-Aldrich). Two daysafter infection, the infected cells were selected with 600 μg/ml of G418(Sigma-Aldrich), 2 μg/ml of puromycin (Sigma-Aldrich) or 1 mg/ml ofzeocin (Invitrogen).

siRNA and Antisense Oligonucleotides

A stealth siRNA duplex oligoribonucleotide targeting Δ133p53 mRNA(Δ133si-1, 5′-UGU UCA CUU GUG CCC UGA CUU UCA A-3′, SEQ ID NO:1), itsscrambled control, and a standard siRNA duplex oligoribonucleotidetargeting Δ133p53 mRNA (Δ133si-2, 5′-CUU GUG CCC UGA CUU UCAA[dT][dT]-3′, SEQ ID NO:2) were synthesized at Invitrogen. The followingantisense 2′-O-methyl oligonucleotides were purchased from IntegratedDNA Technologies (Coralville, Iowa): 5′-AAC AAC CAG CUA AGA CAC UGCCA-3′ (SEQ ID NO:3) for inhibiting miR-34a; and 5′-AAG GCA AGC UGA CCCUGA AGU-3′ (SEQ ID NO:4) as a control, which is complementary to theenhanced green fluorescence protein (EGFP). These siRNA and antisenseoligonucleotides were transfected at the final concentration of 12 nMand 40 nM, respectively, into MRC-5 and WI-38 fibroblasts by using theLipofectamine RNAiMAX transfection reagent (Invitrogen) according to thesupplier's protocol.

Cell Proliferation Assay, Senescence-Associated-β-Galactosidase(SA-β-gal) Staining, Examination of Cellular Replicative Lifespan, andBromo-Deoxyuridine (BrdU) Incorporation Assay

For cell proliferation assay, 2.4×10⁵ cells per well were plated into12-well plates.

These cells were collected and counted daily for a week using ahematocytometer. The experiments were performed at least twice and dataat each time point were in triplicate. For examining cellularreplicative lifespan, the number of cells was counted at each passage,and the number of population doublings (PDL) achieved between passageswas determined by log₂ (number of cells obtained/number of cellsinoculated) (Michishita, E. et al., Mol. Biol. Cell 16:4623 (2005);Pereira-Smith, O. M. et al., Somatic Cell Genet. 7:411 (1981)). SA-β-galstaining was performed as previously described (Dimri, G. P. et al.,Proc. Natl. Acad. Sci. USA 92:9363 (1995)). For BrdU incorporationassay, cells were incubated with 10 μM of BrdU for 24 h. Theincorporated BrdU was detected using an anti-BrdU monoclonal antibody(Amersham Biosciences) and observed with a fluorescent microscope. Thenuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).

Immunoblot Analysis and Immunoprecipitation

Cells were lysed in RIPA buffer [10 mM Tris-HCl, pH 7.5, 150 mM NaCl,0.1% SDS, 0.1% sodium deoxycholate, 1 mM EDTA, 1% NP-40, completeprotease inhibitors (Roche), phosphatase inhibitor cocktail 1 and 2(Sigma)]. Lysates were separated by SDS-PAGE and transferred tonitrocellulose membranes (BIO-RAD) Immunoblot analysis was accomplishedaccording to standard procedures using ECL detection (AmershamBioscience) or SuperSignal West Dura Extended Duration system (PIERCE).

A polyclonal antibody specifically recognizing Δ133p53 (MAP4) was raisedat Moravian Biotechnology (Brno, Czech Republic) in rabbits injectedwith a mixture of peptides MFCQLAKTC (SEQ ID NO:13) and FCQLAKTCP (SEQID NO:14), which were synthesized as Multiple Antigenic Peptide by G.Bloomberg (University of Bristol, Bristol,UK). The other primaryantibodies used were: TLQ40 (Bourdon, J. C. et al., Genes Dev. 19:2122(2005; Murray-Zmijewski, F. et al., Cell Death Differ. 13:962 (2006))for p53β; CM1 (Bourdon, J. C. et al., Genes Dev. 19:2122 (2005);Murray-Zmijewski, F. et al., Cell Death Differ. 13:962 (2006)), DO-12(Chemicon) and DO-1 (Santa Cruz) for p53; H-164 (Santa Cruz) forp21^(WAF1); SMP14 (Santa Cruz) for MDM2; 4A794 (Upstate) for TRF2; M2monoclonal antibody (Sigma) for FLAG tag; AC-15 (Sigma) for β-actin;anti-Myc tag antibody (Invitrogen); anti-ubiquitin ligase Siah-1A (AvivaSystems Biology); and anti-β-catenin mouse monoclonal antibody (BDBiosciences). Horseradish peroxidase-conjugated goat anti-mouse oranti-rabbit antibodies (Santa Cruz) were used as secondary antibodies.

Real-Time qRT-PCR for Quantification of MicroRNA (miRNA) Expression

RNA samples were prepared by using Trizol (Invitrogen). Reversetranscriptase reactions were performed using TagMan miRNA reversetranscription kit (Applied Biosystems, cat. no. 4366596) and amiR-34a-specific primer. The TagMan miRNA assay kit for miR-34a (AppliedBiosystems, cat. no. 4373278) was used according to the supplier'sprotocol. Real-time PCR reactions were performed in triplicate. RNU66(Applied Biosystems, cat. no. 4373382) was used as a control forquantification. Based on Ct (cycle threshold) values from miR-34a andRNU66 detections, normalized miR-34a expression was calculated by usingthe AACt method according to the supplier's protocol (protocol no.4310255B and User Bulletin no. 4303859B found on the world wide web atappliedbiosystems.com/index.cfm).

Measurement of Telomeric 3′ Overhang and Telomere Length

Genomic DNA samples were digested with Hinf I and electrophoresedthrough 0.7% agarose gel. After drying at 25° C. for 30 min in a Bio-Radmodel 583 gel dryer, the gel was hybridized with ³²P-labeled [CCCTAA]₄(SEQ ID NO:5) oligonucleotide as previously described (Miura, N. et al.,Cancer Genet. Cytogenet. 93:56 (1997)), followed by washing and signaldetection using the Typhoon 8600 system (Molecular Dynamics, Sunnyvale,Calif.). The amounts of telomeric 3′ overhangs, normalized with loadedDNA amounts detected with ethidium bromide (EtBr) staining of the gel,were quantitated by using the ImageQuant version 5.2 software (MolecularDynamics). After alkali denaturation (0.5M NaOH/1.5M NaCl) andneutralization (2.5M NaCl/0.5M Tris-HCl, pH 7.5) of the dried gel, thesame procedures were repeated to examine telomere length, which wasindicated as a peak TRF (terminal restriction fragment) length.

Example 2

Expression of p53β and Δ133p53 and p53 Target Genes

The antibodies specific to p53β (TLQ40) (Bourdon, J. C. et al., GenesDev. 19:2122 (2005); Murray-Zmijewski, F. et al., Cell Death Differ.13:962 (2006)) and Δ133p53 (MAP4; see Materials and Methods and FIG. 5)were raised and used to examine the endogenous expression of p53β andΔ133p53 in normal human fibroblast strains (MRC-5 and WI-38) at earlypassage and at replicative senescence (Y and S, respectively, in FIG.1A). While the expression of wt p53 (detected by CM1) showed no changesduring replicative senescence in these fibroblasts, p53β wasspecifically detected when the cells became senescent. In remarkablecontrast, the expression of Δ133p53 was diminished in the senescentcells. The RT-PCR analyses showed neither an increase in the alternativeRNA splicing producing p53β nor a decrease in the usage of the intron 4promoter driving Δ133p53 in senescent fibroblasts (FIG. 6), suggestingthat the induction of p53β and the repression of A133p53 occur at theposttranscriptional levels during replicative senescence. Thesenescence-associated changes in p53β and Δ133p53 coincided with theupregulation of p21^(WAF1) (FIG. 1A), an effector of p53-mediatedcellular senescence (Herbig, U. et al., Mol. Cell 14:501 (2004); Brown,J. P. et al., Science 277:831 (1997)). An increased expression ofmiR-34a, a microRNA (miRNA) that is transcriptionally activated by wtp53 and has an ability to induce cellular senescence when overexpressed(Chang, T. C. et al., Mol. Cell 26:745 (2007); He, L. et al., Nature447:1130 (2007); Raver-Shapira, N. et al., Mol. Cell 26:731 (2007)), wasobserved in MRC-5 and WI-38 when they entered into senescence (FIG. 1B),suggesting that the endogenous expression of miR-34a is involved in thep53-mediated regulation of replicative senescence.

Example 3

Effect of Overexpression of p53β and Δ133p53 on Cell Proliferation andSenescence

The senescence-specific changes in the endogenous p53β and Δ133p53expression prompted us to examine the effects of overexpression of thesep53 isoforms on cell proliferation and senescence. The FLAG-tagged p53βand Δ133p53, as well as wt p53, were retrovirally expressed in theearly-passage human fibroblast strains (FIG. 1C). Similar to wt p53,p53β inhibited cell proliferation (FIG. 1D) and induced cellularsenescence, characterized by the senescence-associated β-galactosidase(SA-β-Gal) activity (FIG. 1E and FIG. 7). The senescence induction byp53β overexpression was associated with the upregulation of the wt p53transcriptional targets, p21^(WAF1) and MDM2 (Rozan, L. M. et al., CellDeath Differ. 14:3 (2007)) (FIG. 1C), confirming that p53β enhances theintrinsic transcriptional activity of p53 as previously described(Bourdon, J. C. et al., Genes Dev. 19:2122 (2005)). p53β also inhibitedcell proliferation and induced cellular senescence in atelomerase-immortalized fibroblast cell line (FIG. 8). However, p53β hadno effects on cell proliferation, cellular senescence or the expressionof p21^(WAF1) and MDM2 in p53-null MDAH041 fibroblasts (Yin, Y. et al.,Cell 70:937 (1992)) (data not shown; also see FIG. 13A below),indicating that p53β co-operates with wt p53 to enhance itssenescence-inducing activity. In marked contrast to wt p53 and p53β, theoverexpression of Δ133p53 accelerated cell proliferation of the normalhuman fibroblasts (FIG. 1D) without inducing cellular senescence (FIG.1E and FIG. 7), and repressed the expression of p21^(WAF1) and MDM2(FIG. 1C).

The biological effects of Δ133p53 were more evident when it wasoverexpressed in the late-passage human fibroblasts, just before thesenescent stage (FIG. 2). In the fibroblast strains MRC-5 and WI-38,whereas the vector control cells underwent senescent growth arrest atonly three or five population doublings (PDLs) after retroviraltransduction, the Δ133p53-overexpressing cells bypassed this normalsenescence point and continued to proliferate for an additional 10 or 15PDLs (FIG. 2A and FIG. 9). The analysis of telomeres revealed that theΔ133p53-induced extension of the replicative lifespan was not due totelomere stabilization: both the overall length of telomeres and theamount of telomeric 3′ overhangs continued to be reduced in theΔ133p53-overexpressing cells (FIG. 2B). The peak length of telomereterminal restriction fragments (TRF) in the Δ133p53-overexpressing MRC-5at the end of the replicative lifespan was reduced down to 4.3 Kbs,which was 1.8-Kb shorter than that in the senescent vector controlcells. The relative amount of 3′ overhangs at the end of the replicativelifespan was also less in the Δ133p53-overexpressing cells than in thevector control cells (37% in the former versus 71% in the latter). Thesedata suggest that the Δ133p53 expression allowed normal human cells tocontinue proliferating beyond the checkpoint defined by a certain levelof telomere length and 3′ overhang amount, which would otherwise lead tocellular senescence (Stewart, S. A. et al., Nat. Genet. 33:492 (2003)).As shown in FIG. 2C, the expression of miR-34a in Δ133p53-overexpressingMRC-5 fibroblasts remained restricted to low levels throughout theirreplicative lifespan. The inhibition of miR-34a expression by anantisense oligonucleotide extended the replicative lifespan in MRC-5fibroblasts (FIG. 2D). These results suggest that the impaired inductionof this p53 target miRNA contributes to the extension of replicativelifespan by Δ133p53.

Example 4 Effect of Inhibition of Δ133p53 on Cell Senescence

To examine the physiological role of Δ133p53 in the regulation ofcellular senescence, the endogenous expression of Δ133p53 was knockeddown by RNA interference in early-passage MRC-5 (FIG. 3) and WI-38fibroblasts (FIG. 10). Two small interfering RNA (siRNA)oligonucleotides (Δ133si-1 and Δ133si-2), which target the sequencesthat are present in Δ133p53 mRNA as 5′ untranslated region but splicedout of wt p53 mRNA as intron 4, efficiently downregulated the endogenousΔ133p53 without affecting wt p53 expression (FIG. 3A and FIG. 10A). Thecells transfected with Δ133si-1 and Δ133si-2, but not those with acontrol scrambled oligonucleotide, underwent a senescent growth arrestuniformly and rapidly (within 7 days), showing the flattened cellmorphology (FIG. 3B and FIG. 10B), the induction of SA-β-Gal activity(FIG. 3B, 3C and FIG. 10B), the attenuation of BrdU incorporation (FIG.3D and FIG. 10C) and the upregulation of p21^(WAF1) (FIG. 3A and FIG.10A). These results indicate that the endogenous expression of Δ133p53is critical to the replicative potential of normal human fibroblasts,and that the downregulation of Δ133p53 plays a causative role in aphysiological induction of cellular senescence.

The continuous cell proliferation beyond the normal senescencecheckpoint with a progressive erosion of telomeres was previouslyobserved in various human cell culture systems, including SV40 large T-and HPV E6/E7-expressing fibroblasts (Harley, C. B. et al., Gerontol.27:375 (1992)). Similar to these precedents, the lifespan extension byΔ133p53 was associated with the impaired expression of p21^(WAF1) (FIG.1C), a cyclin-dependent kinase inhibitor responsible for p53-mediatedcellular senescence (Herbig, U. et al., Mol. Cell 14:501 (2004); Brown,J. P. et al., Science 277:831 (1997)). The repression of miR-34a byΔ133p53 (FIG. 2C) may allow a group of genes for cell cycle progressionto remain expressed (Chang, T. C. et al., Mol. Cell 26:745 (2007)). Itis thus likely that the dysregulated cell cycle progression, even in thepresence of short telomeres, underlay the lifespan extension by Δ133p53.Our results indicate that Δ133p53 might also have an effect ontelomeres.

Example 5 Effect of Inhibition of Δ133p53 on Proteins Involved inTelomere Length

The overexpression of the telomere binding protein, TRF2, in cells withattenuated p53 function reset the senescence setpoint of telomeres to ashorter length and delayed the onset of cellular senescence (Karlseder,J. et al., Science 295:2446 (2002)). Therefore, we investigated whetherΔ133p53 functions in part through the regulation of TRF2 expression. Theupregulation of TRF2 was observed at all of the time points examined inthe two fibroblast strains overexpressing Δ133p53 (FIG. 4A). We alsofound that shRNA knockdown of p53 induced the expression of TRF2 protein(FIG. 4B). Consistently, the small-molecule inhibitor of MDM2(Nutlin-3a) (Buolamwini, J. K. et al., Curr. Cancer Drug Targets 5:57(2005)), which increases p53 stability and transcriptional activity,resulted in a p53-dependent decrease in TRF2 expression (FIG. 11). Thesep53-dependent changes in TRF2 protein expression were inverselycorrelated with the changes in expression of the p53-regulated miR-34a:the shRNA knockdown of p53 and the treatment with Nutlin-3a reduced andinduced miR-34a expression, respectively (FIG. 4C). However, a direct,miR-34a-targeted downregulation of TRF2 protein was unlikely because theretroviral overexpression of miR-34a resulted in no change in TRF2expression (data not shown).

In three fibroblast strains derived from Li-Fraumeni syndrome patients(FIG. 4D; MDAH041, MDAH087 and MDAH178) (Yin, Y. et al.,. Cell 70:937(1992)), the loss of wt p53 allele (−/− and mt/mt) induced or enhancedthe TRF2 expression, providing further evidence for the repression ofTRF2 by wt p53. As shown in FIG. 4E, although Δ133p53 by itself did notaffect the TRF2 expression, it had the ability to cancel the wtp53-mediated downregulation of TRF2. The treatment of human fibroblastswith a proteasome inhibitor MG-132 resulted in an increased TRF2 proteinamount comparable with that in the p53-knocked-down andΔ133p53-expressing cells, while the same treatment did not lead to anadditional increase in these cells (FIG. 4F). The increase in TRF2protein expression by p53 knockdown occurred without a change in TRF2mRNA expression (FIG. 12). These findings indicate that wt p53negatively regulates TRF2 through a proteasomal degradation pathway andthat the inhibition of wt p53 by shRNA knockdown and Δ133p53 expressionstabilizes TRF2, providing a mechanistic, causative link between the p53inactivation and the TRF2 upregulation in human cancers (Nakanishi, K.et al., Clin. Cancer Res. 9:1105 (2003); Nijjar, T. et al., Oncogene24:3369 (2005)).

We did not find a physical interaction between TRF2 and wt p53 in animmunoprecipitation (IP)-Western blot analysis (data not shown) andassumed that wt p53 regulates the TRF2 degradation through itstranscriptional target involved in the proteasome pathway, rather thanthrough a direct association with TRF2. MDM2, an E3 ubiquitin ligasewell known to be transcriptionally upregulated by wt p53 (Rozan, L. M.et al., Cell Death Differ. 14:3 (2007)), was unlikely to be responsiblefor the wt p53-mediated TRF2 degradation, because the p53-dependentdecrease in TRF2 expression occurred even when MDM2 was inactivated byits specific inhibitor Nutlin-3a (FIG. 11). Because TRF2 contains a MybDNA-binding domain at the C-terminus (van Steensel, B. et al., Cell92:401 (1998)), we focused on another E3 ubiquitin ligasetranscriptionally induced by wt p53, Siah-1A (Matsuzawa, S. et al., EMBOJ. 17:2736 (1998)) (FIG. 13A), which causes protein degradation througha Myb DNA-binding domain (Tanikawa, J. et al., J. Biol. Chem. 279:55393(2004)). As shown in FIG. 4G, the accumulation of endogenous TRF2 wasobserved with the inhibition of endogenous Siah-1A activity by thedominant-negative mutant lacking the RING finger domain (Hu, G. et al.,Mol. Cell. Biol. 19:724 (1999)). It was also shown that theoverexpression of Siah-1A activity resulted in the downregulation ofTRF2 (FIG. 13B). These results suggest that Siah-1A is responsible forthe wt p53-induced TRF2 degradation.

The present data and our previous results (Bourdon, J. C. et al., GenesDev. 19:2122 (2005)) suggest that Δ133p53 can inhibit thetranscriptional activity of wt p53. The effects of Δ133p53 in thepresence of wt p53, an in vivo physical interaction of wt p53 andΔ133p53 shown by an IP-Western experiment (data not shown), and the lackof the N-terminal transactivation domain in Δ133p53 all suggest thedominant-negative regulation of the wt p53 function by Δ133p53. Byanalogy to the gain-of-function activity of some mutant p53 proteins(Kastan, M. B. et al., Nat. Cell Biol. 9:489 (2007)), we investigatedwhether Δ133p53 also functions independently of wt p53. When Δ133p53 wasoverexpressed in the Li-Fraumeni syndrome fibroblast MDAH041 null forp53 (homozygous for a frameshift mutation at codon 184) (Yin, Y. et al.,Cell 70:937 (1992)), no significant change in TRF2 expression wasobserved (FIG. 13A). The shRNA knockdown of Δ133p53 in CC1 cells, whichexpress Δ133p53, but not wt p53, due to the homozygous genomic deletion(Horikawa, I. et al., Hum. Mol. Genet. 4:313 (1995)), resulted in nochange in TRF2 expression (FIG. 13C) or cell proliferation (data notshown). Thus, there is no evidence for a gain-of-function activity ofΔ133p53.

Discussion

This study provides a novel mechanistic link among p53, telomeres andcellular senescence in humans by establishing the causative role of p53in the regulation of TRF2, a key component of the telomere-bindingprotein complex (Verdun, R. E. et al., Nature 447:924 (2007)). Our datashow that, in addition to the involvement of p53 in theATM-p53-p21^(WAF1) pathway (Herbig, U. et al., Mol. Cell 14:501 (2004))and the miR-34a-mediated pathway (Chang, T. C. et al., Mol. Cell 26:745(2007); He, L. et al., Nature 447:1130 (2007); Raver-Shapira, N. et al.,Mol. Cell 26:731 (2007)), which could signal DNA damage at telomeres tothe cell cycle control, p53 functions to directly regulate the structureand function of telomeres through the TRF2 regulation. This represents anovel p53 regulation mode of cellular proliferation and senescence. Inagreement with this notion, the TRF2 overexpression extended thecellular replicative lifespan, as previously reported (Karlseder, J. etal., Science 295:2446 (2002)), but to a lesser extent than the Δ133p53overexpression, and the co-overexpression of TRF2 and Δ133p53 had aminimal additional effect to the overexpression of Δ133p53 alone (FIG.4H). In summary, this study indicates that Δ133p53 functions as aphysiological regulator of cellular senescence by modulating multiple,wt p53-regulated signaling pathways to cellular senescence. Given thatTRF2 inhibits the ATM-initiated DNA damage signaling at telomeres(Denchi, E. L. et al., Nature 448:1068 (2007)), our data also suggestthat a feedback loop involving ATM, p53 and TRF2 may amplify thep53-mediated and DNA damage-induced cellular responses.

A novel mechanism for inactivating the tumor suppressor functions of wtp53 was characterized in this work: the inhibition by its own naturalisoform. Similar to the viral oncoproteins, such as HPV E6 and SV40 Tantigen, in in vitro cell transformation models (Harley, C. B. et al.,Gerontol. 27:375 (1992)), Δ133p53 inhibits the wt 53 activity to extendin vitro replicative lifespan of normal human cells. Our preliminaryfindings showed high levels of Δ133p53 expression in some cancer celllines with wild-type p53 retained (Fujita, K. et al., unpublishedobservations), suggesting that Δ133p53 may also play a critical role inhuman carcinogenesis. Even in the absence ofp53 gene mutations, theupregulation of Δ133p53 could counteract the wt p53 activity, allowingthe premalignant cells to bypass the senescence checkpoint and acquireoncogenic mutations during the extended replicative lifespan.

The senescence-associated p53 isoform switching revealed herein providethe basis for a new strategy for the p53-based manipulation of aging andcarcinogenesis. The detection of highly expressed Δ133p53 in the absenceof a p53 mutation in cancer diagnosis identifies cases in which thespecific inhibition of Δ133p53 can activate p53-dependent cellularsenescence, and therefore, will be of great therapeutic value.

Example 6

Δ133p53, p53β, and a p53-Regulated MicroRNA, miR-34a are Regulators ofReplicative Cellular Senescence

The finite replicative potential of normal human cells leads to anirreversible proliferation arrest called replicative cellularsenescence, which constitutes a critical mechanism for tumoursuppression in vivo and may contribute to organismal ageing. p53 plays acentral role in the regulation of replicative senescence. The human p53gene encodes, in addition to full-length p53, several truncated p53isoforms⁵, whose roles are poorly understood. Here, the inventors showthat the p53 isoforms (Δ133p53 and p53β) and a p53-induced microRNA(miR-34a)⁶ are involved in p53-mediated replicative senescence. Aswitching in endogenous protein expression from Δ133p53 to p53β wasassociated with replicative senescence, but not premature senescenceinduced by either oncogene expression or acute telomere dysfunction, innormal human fibroblasts. The endogenous expression of miR-34a was alsoupregulated at replicative senescence. The siRNA-mediated knockdown ofendogenous Δ133p53 induced cellular senescence, which was associatedwith the upregulation of p53 transcriptional targets p21^(WAF1) andPAI-I⁷. Conversely, the antisense inhibition of endogenous miR-34adelayed the onset of replicative senescence. In the overexpressionexperiments, p53β cooperated with full-length p53 to accelerate cellularsenescence, while Δ133p53 extended the cellular replicative lifespanwith the repression of miR-34a, further supporting the roles of the p53isoforms and miR-34a in cellular senescence. It is also discovered thatfreshly isolated, human senescent T lymphocytes (CD8⁺, CD28⁻ andCD57^(+8, 9); and with an increased senescence marker HP1-γ^(10, 11))and colon adenoma tissues with senescence markers, p16^(INK4A) andsenescence-associated β-galactosidase^(12, 13), expressed elevatedlevels of p53β and reduced levels of Δ133p53. Senescence-associated p53isoform switching occurs during both physiologically and pathologicallyinduced senescence, and in various human cell types in vitro and invivo.

Our recent progress provides evidence for the p53 isoform switching inhumans in vivo. We examined senescent CD8+T lymphocytes, which aremarked by CD28−/CD57+ and accumulate as humans age and in HIV (humanimmunodeficiency virus)-infected persons (Effros et al., Immunol. Rev.205: 147-57, 2005; Brenchley et al., Blood 101: 2711-20, 2003) andobserved elevated levels of p53β and reduced levels of Δ133p53 in thesesenescent T cells. Human colon adenomas, premalignant tumors associatedwith accelerated senescence (Kuilman et al., Cell 133: 1019-31, 2008;Collado et al., Nature 436; 642, 2005), also had elevated levels of p53βand reduced levels of Δ133p53. These in vivo results reproduce thefindings from cultured fibroblasts in vitro and indicate a therapeuticapplication of the p53 isoforms.

Normal human somatic cells can undergo only a limited number of celldivisions, eventually reaching an irreversible proliferation arrestcalled replicative cellular senescence^(2, 9). Various cellular stresses(e.g., oncogene activation, oxidative stress and DNA damage) can alsoinduce cellular senescence¹⁻³. Whether replicatively induced orprematurely stress-induced, cellular senescence constitutes a criticalmechanism for tumour suppression in vivo and may contribute toorganismal ageing¹⁻³. The p53 signalling pathway plays a central role inthe regulation of cellular senescence^(2, 3). Although the alternativemRNA splicings and the use of an alternative promoter in the human p53gene produce several truncated p53 isoforms⁴, their regulation andfunction are poorly understood. Here, we examine the expression profilesof two p53 isoforms (p53β and Δ133p53, for which the specific antibodieswere raised; see below) during cellular senescence in vitro and in vivo,their biological activities in regulating cellular senescence, and therole of miR-34a⁵ as a downstream effector of p53-mediated senescence.

The endogenous expression of two major p53 isoforms, p53β and Δ133p53,was examined in normal human fibroblast strains (MRC-5 and WI-38) atearly passage (both strains at passage number 30, Y in FIG. 14 a) and atreplicative senescence (MRC-5 at passage 65 and WI-38 at passage 58, Sin FIG. 14 a) (FIG. 18 a). p5313, detected by the TLQ40 antibody⁵, lacksthe C-terminal oligomerisation domain due to an alternative RNAsplicing⁵; and Δ133p53, detected by the MAP4 antibody (FIG. 19), lacksthe N-terminal transactivation and proline-rich domains due to thetranscription from an alternative promoter in intron 4⁵.

While the expression of full-length p53 (detected by DO-12) showed nochanges during replicative senescence, p53β was specifically detectedwhen the cells became senescent. In remarkable contrast, the expressionof Δ133p53 was markedly diminished in the senescent cells. Theimmunoblot analysis using CM1 antibody showed that p53β and Δ133p53 wereexpressed less abundantly than full-length p53 but still at readilydetectable levels (FIG. 14 a).

Premature senescence induced by oncogenic Ras (FIG. 18 b) or acutetelomere dysfunction (by knockdown of POT1¹⁴ or overexpression of adominant-negative TRF2 mutant¹⁵) was not associated with either inducedp53β or diminished Δ133p53 (FIG. 20), suggesting that the p53 isoformswitching is specific to replicative cellular senescence. The RT-PCRanalyses showed that the p53 isoform switching at replicative senescencewas not due to a change in mRNA expression (FIG. 35).

In addition to the upregulation of p21^(WAF1) (FIG. 14 a), whichmediates p53-induced senescence^(16, 17), the replicatively senescentMRC-5 and WI-38 fibroblasts expressed increased amounts of miR-34a (FIG.14 b), a microRNA that is transcriptionally activated by full-lengthp53^(6, 18) (FIG. 21) and has an ability to induce cellular senescencewhen overexpressed^(6, 19). To examine the role of endogenous miR-34a incellular senescence, an antisense oligonucleotide was developed toknockdown the endogenous expression of miR-34a (FIG. 14 c). Theantisense inhibition of miR-34a in late-passage human fibroblasts (MRC-5at passage 58) extended their replicative lifespan by approximately fivepopulation doublings (PDLs) (FIG. 14 d). The Nutlin-3A-inducedsenescence, which is dependent on the accumulation and activation ofendogenous p53²⁰, was significantly but partially (by approximately 50%)inhibited by the antisense knockdown of endogenous miR-34a (FIG. 14 e).These findings provide the first evidence that the endogenous levels ofmiR-34a, as one of the downstream effectors of p53, plays aphysiological role in the regulation of cellular senescence.

The endogenous expression of Δ133p53 was knocked down by RNAinterference in early-passage WI-38 (FIG. 15) and MRC-5 (FIG. 22). Twosmall interfering RNA (siRNA) oligonucleotides (Δ133si-1 and Δ133si-2),which target the sequences that are present in Δ133p53 mRNA as 5′untranslated region, but spliced out of full-length p53 mRNA as intron4, efficiently downregulated the endogenous Δ133p53 with a minimaleffect on full-length p53 and no induction of p53β (FIG. 15 a and FIG.22 a). The cells transfected with Δ133si-1 and Δ133si-2, but not thosewith a control scrambled oligonucleotide, underwent a senescent growtharrest uniformly and rapidly (within 7 days), showing the flattened cellmorphology (FIG. 15 b, FIG. 22 b), the induction of SA-13-gal activity(FIG. 15 b, 15 c, 22 b), and the attenuation of BrdU incorporation (FIG.15 d, 22 c). These results indicate that the endogenous expression ofΔ133p53 is critical to the replicative potential of normal humanfibroblasts, and that the downregulation of Δ133p53 can play aphysiological role in the induction of cellular senescence. The Δ133p53knockdown-induced senescence was accompanied by the upregulation ofp21^(WAF1) (FIG. 15 a, 22 a) and PAI-1 (plasminogen activatorinhibitor-1) (FIG. 15 e), another p53 target gene responsible for theinduction of replicative senescence', consistent with the activation ofthe full-length p53 function upon a relief from the dominant-negativeinhibition by Δ133p53⁵. Immunoblot analyses of PARP [poly(ADP-ribose)polymerase] or caspase-3 did not show a sign of apoptosis in thesesiRNA-transfected fibroblasts (FIG. 23). Unlike at replicativesenescence, miR-34a was not upregulated at Δ133p53 knockdown-inducedsenescence (FIG. 24), suggesting that some (e.g., 21^(WAF1) and PAI-1),but not all (e.g., miR-34a), p53 target genes respond to an acuteinhibition of Δ133p53 and are sufficient for the full induction ofcellular senescence.

To further examine the effects of the p53 isoforms on cell proliferationand senescence, the FLAG-tagged p53β and Δ133p53, as well as full-lengthp53, were retrovirally expressed in the early-passage human fibroblaststrains (FIG. 16, 25). Similar to full-length p53, p53β inhibited cellproliferation (FIG. 16 a) and induced cellular senescence, characterizedby the senescence-associated β-galactosidase (SA-β-gal) activity (FIG.16 b).

The senescence induction by p53β overexpression was associated with theupregulation of the full-length p53 transcriptional targets, p21^(WAF1)and MDM2²¹ (FIG. 25), confirming that p53β enhances the intrinsictranscriptional activity of p53 as previously described⁵. p53β alsoinhibited cell proliferation and induced cellular senescence in atelomerase-immortalized fibroblast cell line (FIG. 26). However, theoverexpression of p53β had no effect on cell proliferation, cellularsenescence or the expression of p21^(WAF1) and MDM2 in p53-null MDAH041fibroblasts (homozygous for a frameshift mutation at codon 184)²² (datanot shown), indicating that p53β co-operates with full-length p53 toenhance its senescence-inducing activity. In contrast to full-length p53and p53β, the overexpression of Δ133p53 in MRC-5 and WI-38 fibroblastsaccelerated cell proliferation (FIG. 16 a) without inducing cellularsenescence (FIG. 16 b), and repressed the expression of p21^(WAF1) andMDM2 (FIG. 25).

The biological effects of Δ133p53 were more evident when overexpressedin the late-passage human fibroblasts, just before the senescent stage.In MRC-5 and WI-38, whereas the vector control cells underwent senescentgrowth arrest at only one to five PDLs after retroviral transduction,the Δ133p53-overexpressing cells reproducibly bypassed this normalsenescence point and continued to proliferate for six to 15 more PDLs(FIG. 16 c, 16 d, 27). As shown in FIG. 16 e, the expression of miR-34ain Δ133p53-overexpressing MRC-5 remained restricted to low levelsthroughout the replicative lifespan. The Δ133p53-induced extension ofthe replicative lifespan was not due to telomere stabilization; in theΔ133p53-overexpressing cells, both the overall length of telomeres andthe amount of telomeric 3′ overhangs continued to be reduced beyondthose in the senescent vector control cells (FIG. 16 f; compare Δ133p53at day 96 and vector at day 35). Similar to other human cell culturesshowing the lifespan extension with a progressive erosion of telomeres,including SV40 large T- and HPV E6/E7-expressing fibroblasts²³, theextension of replicative lifespan by Δ133p53 is thus attributed to therepression of p21^(WAF1) (FIG. 25), which results in the failure toarrest the cell cycle¹⁶, and the repression of miR-34a (FIG. 16 e),which can allow a group of genes for cell cycle progression to remainexpressed¹⁸.

To investigate whether the p53 isoforms are also involved in cellularsenescence in vivo, CD8⁺ T lymphocytes were freshly isolated fromhealthy donors of age ≧50 yrs and fractionated by flow cytometry usingthe CD28 and CD57 antibodies (FIGS. 34 a, 36 a and 36 b), where CD28⁻and CD57⁺ were the surface markers specific to replicative senescence ofCD8⁺ T lymphocytes⁸′ ⁹. The senescent state of these CD8⁺ T lymphocyteswas confirmed by increased levels of a senescence marker HP1-γ^(10, 11)(FIG. 34 b, FIG. 36 c). The results from three independent donors showedthat Δ133p53 and p53β were down- and up-regulated, respectively, in theorder from CD28⁺ CD57⁻ (non-senescent), CD28⁺CD57⁺, CD28⁻ CD57⁻ to CD28⁻CD57⁺ (most senescent) fractions (FIG. 35 b, 35 c, 37), in vivoreproducing the p53 isoform switching as observed in human fibroblastsin cell culture in vitro. We also examined human colon adenomas, whichare premalignant tumours associated with telomere shortening-inducedreplicative senescence^(24, 25) and oncogene-induced,interleukin-regulated premature senescence^(10, 12, 13). Consistently,we observed positive SA-β-gal staining in adenoma tissues (FIG. 34 d).When normal colon tissues obtained from immediate autopsy²⁶ (n32 9)(Table 1) and 8 pairs of surgically resected, matched non-adenoma andadenoma tissues (Table 2) were compared (FIG. 34 e, FIG. 28), theexpression of p16^(INK4A), an in vivo senescence marker²⁷, wassignificantly more abundant in colon adenomas than in non-adenomas ornormal colon tissues, as reported previously^(12, 28.) As expected fromthis increase in senescence, colon adenoma tissues expressed elevatedlevels of p53β and reduced levels of Δ133p53 compared with non-adenomaand normal colon tissues. The increased expression of p16^(INK4A) andp53β and the decreased expression of Δ133p53 in adenoma tissues werealso observed in the paired analysis of the 8 cases of matchednon-adenoma and adenoma tissues (FIG. 34 f). These results suggest thatthe p53 isoform switching occurs not only in cultured cells in vitro butalso in humans in vivo, during both physiologically and pathologicallyinduced senescence (T lymphocytes in the elderly and colon adenomas,respectively), and in cells of different origins (mesenchymal,hematopoietic and epithelial origins).

We also examined 29 cases of matched colon carcinoma and non-carcinomatissues (Table 3) for Δ133p53 and p53β expression (FIG. 31). In contrastto colon adenomas, colon carcinoma tissues (FIG. 17 b, bars “Ca”) didnot show the senescence-associated p53 isoform expression signature,with Δ133p53 increased and p53β decreased back to similar levels tothose in normal colons and non-adenoma tissues. Although the adjacentnon-carcinoma tissues (FIG. 17 b, bars “Non-ca”) expressed significantlyelevated levels of p53β, which were comparable to those in adenomas, itsbiological importance is currently unknown. When colon carcinoma tissueswere compared among clinical stages (FIG. 17 c), the stage I carcinomasalready failed to maintain the characteristics of adenomas, showingsignificantly increased Δ133p53 and decreased p53β compared withadenomas. These results show that the senescence-associated p53 isoformexpression signature becomes lost at an early stage of malignantprogression. The loss of the signature may signal an escape from thesenescence barrier observed in premalignant tumors^(1, 3, 8, 20, 23). Afurther significant increase in Δ133p53 from stage Ito II and a furtherdecrease in p53β from stage II to III (FIG. 17 c) suggest that these p53isoforms may also play a role during stage progression of coloncarcinoma. The biological relevance of the function of Δ133p53 in coloncarcinogenesis was further substantiated by the subgroup analysis basedon p53 status, which was determined by p53 and MDM2 immunohistochemicalstaining of carcinoma tissues^(24, 25) (Table 3). The expression levelsof Δ133p53 were significantly higher in carcinoma tissues than innon-carcinoma tissues in the cases assumed to be ‘wild-type’ p53, butnot in the cases assumed to be ‘mutant’ p53 (FIG. 32 and Table 4). Thisfinding agrees with our in vitro data showing the ability of Δ133p53 toinhibit the wild-type p53 function (FIGS. 15 a, 15 e, 16 e, and 25) andsuggests that elevated levels of Δ133p53 may replace p53 gene mutationsin colon carcinogenesis in vivo.

Interleukin-8 (IL-8) was upregulated in colon adenoma tissues comparedwith adjacent non-adenoma tissues (FIG. 33), reproducing the recentreport by Kuilman et al.⁸ and further confirming the senescence statusof the adenoma samples used in this study. The IL-8 signalling pathwayseems involved in both replicative senescence and oncogene-inducedsenescence in a p53-dependent manner²³, which are observed in colonadenomass^(8, 18-20). However, it is unlikely that thiscytokine-mediated mechanism for senescence primarily regulates, or isregulated by, the senescence-associated expression signature of the p53isoforms, because colon carcinoma tissues without such signature (FIG.17 b) still expressed remarkably increased levels of IL-8 (FIG. 33), asreported²⁶, and adjacent non-carcinoma tissues with elevated p53β (FIG.17 b) showed no increase in IL-8 expression (FIG. 33). Considering ourin vitro observation that the senescence-associated p53 isoformexpression signature is specific to replicative senescence (FIG. 14 aand FIG. 20), a full malignant conversion from adenoma to carcinoma mayrequire overcoming the senescence barriers by both p53 isoform-dependent(i.e., replicative senescence) and -independent (e.g., oncogene-induced,interleukin-regulated senescence) mechanisms.

In summary, based on the expression and functional analyses ofendogenous proteins, which were supported by the overexpressionexperiments, this study provides the first evidence for thephysiological regulation of replicative cellular senescence by naturalp53 isoforms. The data also establish the endogenous miR-34a as adownstream effector in the p53-regulated signalling pathways to cellularsenescence. Although the exact mechanism of the senescence-associatedp53 isoform switching still remains to be determined, we found thatΔ133p53, unlike p53β and full-length p53, does not accumulate in thepresence of a proteasome inhibitor MG-132 (FIG. 38), suggesting that thedifferential dynamics of protein turnover may be involved. With theevidence for the p53 isoform switching in vivo in both healthy anddisease conditions, this study provides a new p53-based,senescence-mediated strategy for the manipulation of ageing andcarcinogenesis processes in vivo²⁻⁴.

Methods

Retroviral vector transduction was performed essentially as previouslydescribed^(14, 29). Transfection of siRNA and antisense oligonucleotidesused the Lipofectamine RNAiMAX transfection reagent (Invitrogen,Carlsbad, Calif.). Cell proliferation, replicative lifespan andsenescence assays were essentially as described^(14, 20, 29). Forimmunoblot analyses, preparation of protein lysates from cells ortissues, SDS-PAGE, transfer to nitrocellulose or PVDF membranes,incubation with antibodies, and signal detection followed the standardprocedures. The real-time qRT-PCR for miR-34a expression was performedusing the reagents from Applied Biosystems (Foster City, Calif.),essentially as described⁶. To analyze telomeric 3′ overhang and telomerelength, in-gel Southern hybridization with ³²P-labeled [CCCTAA]₄ (SEQ IDNO:5) oligonucleotide, under native and denatured conditions, wasperformed as previously described¹⁴. Fluorescence-activated cell sorting(FACS) of human CD8⁺ T lymphocytes based on CD28 and CD57 expressionpatterns essentially followed Brenchley et al.⁸. Human tissues werecollected with approval from the Institutional Review Board of theNational Institutes of Health.

Cells. CC1, a human choriocarcinoma cell line expressing Δ133p53 due tothe genomic rearrangement deleting the exons 2, 3 and 4³¹, was a giftfrom Dr. Mitsuo Oshimura (Tottori University, Japan). Normal humanfibroblast strains (MRC-5 and WI-38), H1299, RKO and 293T were obtainedfrom American Type Culture Collection (Manassas, Va.). hTERT/NHF, anhTERT (human telomerase reverse transcriptase)-immortalized humanfibroblast cell line, was previously described³². MDAH041 was kindlyprovided by Dr. Michael Tainsky (Case Western Reserve University,Cleveland, Ohio). The treatment with Nutlin-3A was as described²⁰.

Plasmid constructs. To generate the retroviral expression vectors ofhuman p53 isoforms, full-length p53, FLAG-tagged p53β and FLAG-taggedΔ133p53 were PCR-amplified using pSVrp53, pSVp53β and pSVDNp53⁵,respectively, as the templates, and then inserted into Not I and Eco RIsites of pQCXIN vector (BD Biosciences, San Jose, Calif.). Theseconstructs were verified by DNA sequencing. The retroviral constructpLPC-Myc-TRF2^(ΔBΔM) was a gift from Dr. Titia de Lange (RockefellerUniversity, N.Y.). The retroviral expression vector for H-RasV12 was agift from Dr. Manuel Serrano (Spanish National Cancer Research Center).The shRNA knockdown vectors targeting p53 and POT1 were previouslydescribed¹⁴.

Retroviral vector transduction. The retroviral constructs weretransfected into Phoenix packaging cells (Orbigen, Inc., San Diego,Calif.) using Lipofectamin 2000 (Invitrogen, Carlsbad, Calif.). Vectorsupernatants were collected 48 h after transfection and used to infectcells in the presence of 8 μg/ml polybrene (Sigma-Aldrich, St. Louis,Mo.). Two days after infection, the infected cells were selected with600 μg/ml of G418 (Sigma-Aldrich), 2 μg/ml of puromycin (Sigma-Aldrich)or 1 mg/ml of zeocin (Invitrogen).

siRNA and antisense oligonucleotides. A stealth siRNA duplexoligoribonucleotide targeting Δ133p53 mRNA (Δ133si-1, 5′-UGU UCA CUU GUGCCC UGA CUU UCA A-3′, SEQ ID NO:1), its scrambled control, and astandard siRNA duplex oligoribonucleotide targeting Δ133p53 mRNA(Δ133si-2, 5′-CUU GUG CCC UGA CUU UCA A[dT][dT]-3′, SEQ ID NO:2) weresynthesized at Invitrogen. The following antisense 2′-O-methyloligonucleotides were purchased from Integrated DNA Technologies(Coralville, Iowa): 5′-AAC AAC CAG CUA AGA CAC UGC CA-3′, SEQ ID NO:3,for inhibiting miR-34a; and 5′-AAG GCA AGC UGA CCC UGA AGU-3′, SEQ IDNO:4, as a control, which is complementary to the enhanced greenfluorescence protein (EGFP). The siRNA and antisense oligonucleotideswere transfected at the final concentration of 12 nM and 40 nM,respectively, into MRC-5 and WI-3 8 fibroblasts by using theLipofectamine RNAiMAX transfection reagent (Invitrogen) according to thesupplier's protocol.

Cell proliferation assay, senescence-associated-β-galactosidase(SA-β-gal) staining, examination of cellular replicative lifespan, andbromo-deoxyuridine (BrdU) incorporation assay. For cell proliferationassay, 2.4×10⁵ cells per well were plated into 12-well plates. Thesecells were collected and counted daily for a week using ahematocytometer. The experiments were performed at least twice and dataat each time point were in triplicate. For examining cellularreplicative lifespan, the number of cells was counted at each passage,and the number of population doublings (PDL) achieved between passageswas determined by log₂ (number of cells obtained/number of cellsinoculated)²⁹. SA-β-gal staining was performed as previouslydescribed³³. For BrdU incorporation assay, cells were incubated with 10μM of BrdU for 24 h. The incorporated BrdU was detected using ananti-BrdU monoclonal antibody (Amersham Biosciences) and observed with afluorescent microscope. The nuclei were counterstained with4′,6-diamidino-2-phenylindole (DAPI).

Immunoblot analysis. Cells or tissues were lysed in RIPA buffer [10 mMTris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1 mMEDTA, 1% NP-40, complete protease inhibitors (Roche, Indianapolis,Ind.), phoshatase inhibitor cocktail 1 and 2 (Sigma-Aldrich)]. SDS-PAGE,transfer to nitrocellulose or PVDF membranes (Bio-Rad, Hercules,Calif.), incubation with antibodies, and signal detection followed thestandard procedures using ECL detection (Amersham Biosciences,Piscataway, N.J.) or SuperSignal West Dura Extended Duration system(Pierce Biotechnology, Rockford, Ill.). The quantitative analysis of theimmunoblot data was performed using the ImageJ 1.40g software (on theworld wide web at rsb.info.nih.gov/ij/).

Antibodies. A polyclonal antibody specifically recognizing Δ133p53(MAP4) was raised at Moravian Biotechnology (Brno, Czech Republic) inrabbits injected with a mixture of peptides, MFCQLAKTC (SEQ ID NO:13)and FCQLAKTCP (SEQ ID NO: 14), which were synthesized as MultipleAntigenic Peptide by Dr. G. Bloomberg (University of Bristol, Bristol,UK). The other primary antibodies used were: TLQ40⁵ for p53β; CM1⁵,DO-12 (Millipore, Billerica, Mass.) and DO-1 (Santa Cruz Biotechnology,Santa Cruz, Calif.) for p53; EA10 (Carbiochem, San Diego, Calif.) forp21^(WAF1); SMP14 (Santa Cruz Biotechnology) for MDM2; 8G10 (CellSignaling Technology, Danvers, Mass.) for caspase-3; M2 monoclonalantibody (Sigma-Aldrich) for FLAG tag; AC-15 (Sigma-Aldrich) for13-actin; MAB3450 (Chemicon International, Temecula, Calif.) for HP1-γ;anti-phospho-p53 (Ser15) (Cell Signaling Technology); anti-PARP (CellSignaling Technology); FITC-conjugated anti-CD8 (BD Biosciences,Franklin Lakes, N.J.); APC-conjugated anti-CD28 (BD Biosciences); andPE-conjugated anti-CD57 (Abcam, Cambridge, Mass.). Horseradishperoxidase-conjugated goat anti-mouse or anti-rabbit antibodies (SantaCruz Biotechnology) were used as secondary antibodies in immunoblots.

Real-time qRT-PCR for quantification of microRNA expression. RNA sampleswere prepared by using Trizol (Invitrogen). Reverse transcriptasereactions were performed using TaqMan microRNA reverse transcription kit(Applied Biosystems, cat. no. 4366596) and a miR-34a-specific primer.The TaqMan microRNA assay kit for miR-34a (Applied Biosystems, cat. no.4373278) was used according to the supplier's protocol. Real-time PCRreactions were performed in triplicate. RNU66 (Applied Biosystems, cat.no. 4373382) was used as a control for quantification. Based on Ct(cycle threshold) values from miR-34a and RNU66 detections, normalizedmiR-34a expression was calculated by using the AACt method according tothe supplier's protocol (protocol no. 4310255B and User Bulletin no.4303859B found on the world wide web atappliedbiosystems.com/index.cfm).

Real-time qRT-PCR for PAI-1, IL-8 and IL-8R. For quantitativemeasurement of PAI-1 mRNA, the SYBR Green PCR Master Mix (AppliedBiosystems) was used with the following primers: 5′-CTC CTG GTT CTG CCCAAG T-3′ (SEQ ID NO:15) and 5′-CAG GTT CTC TAG GGG CTT CC-3′ (SEQ IDNO:16) for PAI-1; and 5′-TTC TGG CCT GGA GGC TAT C-3′ (SEQ ID NO:17) and5′-TCA GGA AAT TTG ACT TTC CAT TC-3′ (SEQ ID NO:18) for β-2microglobulin as an internal control. For IL-8 and IL-8R, the TaqmanUniversal PCR Master Mix (Applied Biosystems) was used with thefollowing sets of probe and primers purchased from Applied Biosystems:IL-8 (catalog # 4331182, Hs00174103_m1); IL-8R (catalog # 4331182,Hs001174304_m1); and 18S endogenous control (catalog # 4319413).

Measurement of telomeric 3′ overhang and telomere length. Genomic DNAsamples were digested with Hinf I and electrophoresed through 0.7%agarose gel. After drying at 25° C. for 30 min in a Bio-Rad model 583gel dryer, the gel was hybridized with ³²P-labeled [CCCTAA]₄ (SEQ IDNO:5) oligonucleotide as previously described³⁴, followed by washing andsignal detection using the Typhoon 8600 system (Molecular Dynamics,Sunnyvale, Cailf.). The amounts of telomeric 3′ overhangs, normalizedwith loaded DNA amounts detected with ethidium bromide (EtBr) stainingof the gel, were quantitated by using the ImageQuant version 5.2software (Molecular Dynamics). After alkali denaturation (0.5M NaOH/1.5MNaCl) and neutralization (2.5M NaCl1/0.5M Tris-HCl, pH 7.5) of the driedgel, the same procedures were repeated to examine telomere length, whichwas indicated as a peak TRF (terminal restriction fragment) length.

T Cell Sorting. Peripheral blood mononuclear cells from healthyvolunteers were isolated using Histopaque-1077 (Sigma-Aldrich). Theanti-CD57 (PE-conjugated), anti-CD8 (FITC-conjugated) and anti-CD28(APC-conjugated) monoclonal antibodies were added at saturatingconcentrations and the cells were incubated for 30 min on ice and washedtwice, then resuspended at a concentration of 20×10⁶ cells/ml. Thefollowing cell fractions were sorted using the FACSAria cell-sortingsystem (BD Biosciences): CD8+/CD28+/CD57−, CD8+/CD28+/CD57+,CD8+/CD28−/CD57−, CD8+/CD28-/CD57+. Viability (>99%) was determined bygating on 7-AAD-negative cells. Purities of sorted cells were determinedon at least 5000 events and analyzed using FlowJo software (Tree Star,Ashland, Oreg.).

Human colon tissues. Normal colon tissues were obtained from immediateautopsy at Baltimore area hospitals in Maryland²⁶. Pairs of colonadenoma and adjacent non-adenoma tissues were from the University ofMaryland Medical Center and the Cooperative Human Tissue Network. Alltissues were flash frozen immediately after resected. Tumorhistopathology was classified according to the World Health OrganizationClassification of Tumor system. This study was approved by theInstitutional Review Board of the National Institutes of Health. Tables1 and 2 summarize information on tissue samples used in this study.

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TABLE 1 Information on normal colon samples obtained from immediateautopsy. Case number Age Gender Cause of death 1 25 Male Gun shot wound2 29 Male Gun shot wound 3 16 Female Motor vehicle accident (closed headinjury) 4 28 Male Closed head injury 5 23 Female Motor vehicle accident(closed head injury) 6 52 Female Motor vehicle accident 7 76 FemaleMotor vehicle accident 8 20 Male Motor vehicle accident 9 19 Female Gunshot wound

TABLE 2 Information on 8 pairs of colon adenoma and non-adenoma samples.Case number Age Gender Histopathological diagnosis 1 62 Male Tubularadenoma 2 64 Female Tubular adenoma 3 87 Female Villous adenoma 4 84Male Villous adenoma 5 78 Male Tubulovillous adenoma 6 66 Male Tubularadenoma 7 79 Male Villous adenoma 8 78 Male Tubulovillous adenoma

TABLE 3 Information on 29 cases of colon carcinoma. Survival Case*Gender Age Stage p53 status** Histology (months) 10167 M 55 I wild-typeadeno 154.0 10186 F 70 III mutant adeno 153.6 10212 F 66 II wild-typemucinous 144.3 10515 M 53 III mutant adeno 61.9 11148 F 63 II wild-typeadeno 26.6 11157 M 73 II wild-type adeno 130.1 11275 M 76 II wild-typeadeno 90.4 11692 M 58 III mutant adeno 112.3 11731 M 59 III mutantmucinous 18.4 11854 M 70 III n.d.*** adeno 18.8 11873 M 72 II wild-typeadeno 106.7 11918 M 59 II wild-type adeno 104.9 12004 M 51 III mutantadeno 102.1 12031 M 50 II wild-type adeno 38.9 12051 M 70 II wild-typeadeno 79.1 12076 M 76 II mutant adeno 100.1 12124 M 60 III mutant adeno98.6 12158 M 70 III mutant mucinous 97.9 12163 M 53 III Mutant adeno 5.912169 M 67 II wild-type adeno 97.2 12375 F 66 III wild-type mucinous92.2 12879 M 80 I mutant adeno 62.8 12892 M 69 I wild-type adeno 79.913201 F 60 I mutant adeno 72.4 13547 M 69 I wild-type adeno 55.5 13799 M44 II mutant adeno 61.2 14278 M 59 I mutant mucinous 54.1 14554 M 59 Imutant adeno 50.1 15059 M 67 I wild-type adeno 43.5 *Schetter et al.,JAMA 299: 425-436, 2008. **p53 status was assumed to be ‘wild-type’ or‘mutant’ by immunohistochemical staining of p53 and MDM2 (Costa et al.,J. Pathol. 176: 45-53, 1995; Nenutil et al., J. Pathol. 207: 251-259,2005). ***Not determined.

TABLE 4 Δ133p53 and p53β expression in p53 ‘wild-type’ and ‘mutant’cases of colon carcinoma. Δ133p5 p53 Case- Non-caCarcinomaNon-caCarcinoma p53 ‘wild-type’ 10167 - I 0.0285 0.2276 0.3299 0.000412892 - I 0.1376 0.0892 0.1432 0.0190 13547 - I 0.4270 0.1329 0.01460.0604 15059 - I 0.0816 0.2083 0.1946 0.0398 10212 - II 0.0302 0.15290.1059 0.0004 11148 - II 0.1458 0.1007 0.0809 0.1596 11157 - II 0.31050.9175 0.0453 0.0269 11275 - II 0.0986 0.4103 0.0968 0.0061 11873 - II0.3557 0.7519 0.0035 0.0077 11918 - II 0.0885 0.4647 0.1268 0.000412031 - II 0.4436 0.5961 0.0004 0.0004 12051 - II 0.2774 0.0122 0.12130.0004 12169 - II 0.1679 0.6279 0.0154 0.0004 12375 - III 0.0944 0.21260.0897 0.0776 p53 ‘mutant’ 12879 - I 0.2421 0.0033 0.0156 0.0004 13201 -I 0.1807 0.3560 0.1160 0.0304 14278 - I 0.3356 0.2461 0.0395 0.077914554 - I 0.1567 0.2301 0.1226 0.0485 12076 - II 0.3786 0.3812 0.09320.1173 13799 - II 0.0033 0.3206 0.0308 0.0388 10186 - III 0.4134 0.03960.3002 0.0004 10515 - III 0.1003 0.0033 0.0215 0.0004 11692 - III 0.63770.4520 0.0146 0.0088 11731 - III 0.1403 0.0737 0.0166 0.0249 12004 - III0.2440 0.2460 0.0744 0.0019 12124 - III 0.3315 0.5139 0.0769 0.000412158 - III 0.2558 0.5446 0.4359 0.0267 12163 - III 0.2377 0.5289 0.15120.0004

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Sequence Listing SEQ ID NO: 1 5′-UGU UCA CUU GUG CCC UGA CUU UCA A-3′SEQ ID NO: 2 5′-CUU GUG CCC UGA CUU UCA A[dT][dT]-3′ SEQ ID NO: 3 5′-AACAAC CAG CUA AGA CAC UGC CA-3′ SEQ ID NO: 4 5′-AAG GCA AGC UGA CCC UGAAGU-3′

What is claimed is:
 1. A method of extending the replicative lifespan ofa cell by inhibiting cell senescence, the method comprising the step ofcontacting the cell with an agent that inhibits the function orexpression of p53β, thereby inhibiting cell senescence and extending thereplicative lifespan of the cell.
 2. The method of claim 1, wherein theagent is an siRNA or a ribozyme.
 3. The method of claim 1, wherein theagent is an shRNA.
 4. The method of claim 1, wherein the cell is a cellsuitable for tissue regeneration that has a finite number of celldivisions.
 5. The method of claim 4, wherein the method furthercomprises culturing the cell to obtain a cell population for tissueregeneration.
 6. The method of claim 1, wherein the cell is susceptibleto a degenerative disease.
 7. The method of claim 1, wherein the cell isa T cell.
 8. A method of extending the replicative lifespan of a cell byinhibiting cell senescence, the method comprising the step of contactingthe cell with an agent that inhibits the function or expression ofmiR-34a, thereby inhibiting cell senescence and extending thereplicative lifespan of the cell.
 9. The method of claim 8, wherein theagent is an antisense oligonucleotide.
 10. The method of claim 8,wherein the cell is a T cell.
 11. A method of promoting senescence in acell, the method comprising the step of contacting the cell with anagent that activates the function or expression of p53β, therebypromoting cell senescence.
 12. The method of claim 11, wherein the cellis a cancer cell.
 13. The method of claim 11, wherein the agentcomprises a polynucleotide encoding p53β.
 14. The method of claim 11,wherein the agent comprises an expression cassette comprising apolynucleotide sequence encoding p53β.